Methods for regulating gene expression

ABSTRACT

A method for regulating expression of a tet operator-linked gene in a cell of a subject is disclosed. In one embodiment, the method involves introducing into the cell a nucleic acid molecule encoding a tetracycline-controllable transactivator (tTA), the tTA comprising a Tet repressor operably linked to a polypeptide which directly or indirectly activates transcription in eucaryotic cells; and modulating the concentration of a tetracycline, or analogue thereof, in the subject. Alternatively, in another embodiment, the method involves obtaining the cell from the subject, introducing into the cell a first nucleic acid molecule which operatively links a gene to at least one tet operator sequence, introducing into the cell a second nucleic acid molecule encoding a tTA, to form a modified cell, administering the modified cell to the subject, and modulating the concentration of a tetracycline, or analogue thereof, in the subject. The first and second nucleic acid molecule can be within a single molecule (e.g., in the same vector) or on separate molecules.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of application Ser.No. 08/260,452, filed Jun. 14, 1994, which is a continuation-in-part ofapplication Ser. No. 08/076,327, filed Jun. 14, 1993, now abandoned, theentire contents of each of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The study of gene function in complex genetic environments suchas eucaryotic cells would greatly profit from systems that would allowstringent control of the expression of individual genes. Ideally, suchsystems would not only mediate an “on/off” status for gene expressionbut would also permit limited expression at a defined level.

[0003] Attempts to control gene activity by various inducible eucaryoticpromoters responsive to, for example, heavy metal ions (Mayo et al.,Cell 29:99-108 (1982); Brinster et al., Nature (London) 296:39-42(1982); Searle et al., Nouer, L., CRC Boca Raton, Fla. (1991), pp.167-220), or hormones (Lee et al., Nature (London) 294:228-232 (1981);Hynes et al., Proc. Natl. Acad. Sci. USA 78:2038-2042 (1981); Klock etal., Nature (London) 329:734-736 (1987); Israel & Kaufman, Nucleic AcidsRes. 17:2589-2604 (1989)) have generally suffered from leakiness of theinactive state (e.g., the metallothionein promoter (Mayo et al., Cell29:99-108 (1982)) or from pleiotropic effects caused by the inducingprinciples themselves, such as elevated temperature or glucocorticoidhormone action (Lee et al., Proc. Natl. Acad. Sci, USA 85:1204-1208(1988)).

[0004] In search of regulatory systems that do not rely on endogenouscontrol elements, several groups have demonstrated that the lacrepressor/operator inducer system of Escherichia coli functions ineucaryotic cells. Three approaches have been described: (i) preventionof transcription initiation by properly placed lac operators at promotersites (Hu & Davidson, Cell 48:555-566 (1987); Brown et al., Cell49:603-612 (1987); Figge et al., Cell 52:713-722 (1988); Fuerst et al.,Proc. Natl. Acad. Sci. USA 86:2549-2553 (1989); Deutschle et al., Proc.Natl. Acad. Sci. USA 86:5400-5405 (1989)), (ii) blockage of transcribingRNA polymerase II during elongation by a lac repressor/operator complex(lac R/O; Deutschle et al., Science 248:480-483 (1990)), and (iii)activation of a promoter responsive to a fusion between lacR and theactivating domain of virion protein 16 (VP16) of herpes simplex virus(HSV) (Labow et al., Mol. Cell. Biol. 10:3343-3356 (1990); Baim et al.,Proc. Natl. Acad. Sci. USA 88:5072-5076 (1991)).

[0005] At present, however, the utility of the lacR/O-based systems ineucaryotic cells is limited since the inducerisopropyl.β-D-thiogalactopyranoside (IPTG), despite its rapid uptake andintracellular stability (Wyborski & Short, NucleicAcids Res.19:4647-4653), acts rather slowly and inefficiently, resulting in onlymoderate induction. Nevertheless, an interesting conditional mutant of alacR-VP16 fusion has been described (Baim et al., Proc. Natl. Acad. Sci.USA 88:5072-5076 (1991)). It activates a minimal promoter ˜1000-fold atelevated temperatures in the presence of IPTG. The temperaturedependence and the inherent IPTG-related problems, however, may alsolimit this approach.

SUMMARY OF THE INVENTION

[0006] This invention features a system for regulating expression ofeucaryotic genes using components of the Tet repressor/operator/ inducersystem of prokaryotes. In the system of the invention, transcription ofa nucleotide sequence operably linked to at least one tet operatorsequence is stimulated by a tetracycline (Tc)-controllabletranscriptional activator fusion protein (referred to herein as tTA).The tTA is comprised of two polypeptides. The first polypeptide is a Tetrepressor (TetR; e.g., a Tn10-derived TetR), which binds to tet operatorsequences in the absence but not the presence of Tc. The secondpolypeptide directly or indirectly activates transcription in eucaryoticcells. For example, the second polypeptide can be a transcriptionalactivation domain from herpes simplex virus virion protein 16 or anothertranscriptional activating domain, e.g. acidic, proline-rich,serine/threonine-rich, glutamine-rich. Alternatively, the secondpolypeptide can be a domain (e.g., a dimerization domain) which recruitsa transcriptional activator (e.g., an endogenous transcriptionalactivator) to interact with the tTA fusion protein by a protein-proteininteraction (e.g., a non-covalent interaction). In the absence of Tc ora Tc analogue, transcription of a gene operably linked to atTA-responsive promoter (typically comprising at least one tet operatorsequence and a minimal promoter) is stimulated by a tTA of theinvention, whereas in the presence of Tc or a Tc analogue, transcriptionof the gene linked to the tTA-responsive promoter is not stimulated bythe tTA.

[0007] As described herein, this system functions effectively intransgenic animals. Accordingly, the invention provides atetracycline-controllable regulatory system for modulating geneexpression in transgenic animals. Additionally, the invention providestargeting vectors for homologous recombination that enable thecomponents of the regulatory system to be integrated at a predeterminedlocation in the genome of a host cell or animal.

[0008] This embodiment of the invention is able to solve a longstandingproblem in the field generally described as gene targeting or gene knockout. Constitutive disruption of certain genes has been found to producelethal mutations resulting in death of homozygous embryos, e.g., asdescribed for the knock out of the RB gene (Jacks, T. et al. (1992)Nature 359:295-300). This problem precludes the development of “knockout” animals for many genes of interest. The regulatory system of theinvention can be applied to overcome this problem. DNA encoding a tTA ofthe invention can be integrated within a gene of interest such thatexpression of the tTA is controlled by the endogenous regulatoryelements of the gene of interest (e.g., the tTA is expressed spatiallyand temporally in a manner similar to the gene of interest). The gene ofinterest is then operably linked to at least one tet operator sequence(either at its endogenous site by homologous recombination or a secondcopy of the gene of interest, linked to tet operator(s), can beintegrated at another site). Expression of the tet-operator linked geneis thus placed under the control of the tTA, whose pattern of expressionmimics that of the gene of interest. In the absence of Tc, expression ofthe tet operator-linked gene of interest is stimulated by the tTA andthe animal develops like a nonmutated wildtype animal. Then, at aparticular stage of development, expression of the gene of interest canbe switched off by raising the level of Tc (or a Tc analogue) in thecirculation and the tissues of the animal by feeding or injecting Tc (ora Tc analogue) to the animal, thereby inhibiting the activity of the tTAand transcription of the gene of interest.

[0009] This method is generally referred to herein as a “conditionalknockout”.

[0010] Accordingly, one aspect of the invention relates to targetingvectors for homologous recombination. In one embodiment, the inventionprovides an isolated DNA molecule for integrating a polynucleotidesequence encoding a tetracycline-controllable transactivator (tTA) at apredetermined location in a second target DNA molecule. In this DNAmolecule, a polynucleotide sequence encoding a tTA is flanked at 5′ and3′ ends by additional polynucleotide sequences of sufficient length forhomologous recombination between the DNA molecule and the second targetDNA molecule at a predetermined location. Typically, the target DNAmolecule into which the tTA-coding sequences are integrated is a gene ofinterest, or regulatory region thereof, in a eucaryotic chromosome in ahost cell. For example, tTA-coding sequences can be inserted into a genewithin a yeast, fungal, insect or mammalian cell. Additionally,tTA-coding sequences can be inserted into a viral gene present within ahost cell, e.g. a baculovirus gene present in insect host cell. In apreferred embodiment, integration of the tTA-encoding sequences into apredetermined location in a gene of interest (by homologousrecombination) places the tTA-coding sequences under the control ofregulatory elements of the gene of interest (e.g., 5′ flankingregulatory elements), such that the tTA is expressed in a spatial andtemporal manner similar to the gene of interest. In another embodimentof the targeting vector for homologous recombination, the isolated DNAmolecule permits integration of a polynucleotide sequence encoding botha tTA and a tTA-responsive promoter within a predetermined gene ofinterest in a second target DNA molecule (a “single hit vector”,schematically illustrated in FIG. 13A-B). This molecule includes: 1) afirst polynucleotide sequence comprising a 5′ flanking regulatory regionof the gene of interest, operably linked to 2) a second polynucleotidesequence encoding a tTA; and 3) a third polynucleotide sequencecomprising a tTA-responsive promoter, operably linked to: 4) a fourthpolynucleotide sequence comprising at least a portion of a coding regionof the gene of interest. The first and fourth polynucleotide sequencesare of sufficient length for homologous recombination between the DNAmolecule and the gene of interest such that expression of the tTA iscontrolled by 5′ regulatory elements of the gene of interest andexpression of the gene of interest is controlled by the tTA-responsivepromoter (i.e., upon binding of the tTA to the tTA-responsive promoter,expression of the gene of interest is stimulated). This targeting vectorcan also include a polynucleotide sequence encoding a selectable markeroperably linked to a regulatory sequence. Typically, the selectablemarker expression unit is located between the tTA-encoding sequence(i.e., the second polynucleotide sequence described above) and thetTA-responsive promoter (i.e., the third polynucleotide sequencedescribed above). Additionally or alternatively, this targeting vectorcan also include a sequence, typically located upstream (i.e., 5′) ofthe tTA-responsive promoter (e.g., between the selectable markerexpression unit and the tTA responsive promoter) which terminatestranscription or otherwise insulated downstream elements from theeffects of upstream regulatory elements. The tTA-responsive promotertypically includes a minimal promoter operably linked to at least onetet operator sequence. The minimal promoter is derived, for example,from a cytomegalovirus immediate is early gene promoter or a herpessimplex virus thymidine kinase gene promoter.

[0011] Another aspect of the invention relates to eucaryotic host cellscontaining a DNA molecule encoding a tTA integrated at a predeterminedlocation in a second target DNA molecule (e.g., a gene of interest) inthe host cell. Such a eucaryotic host cell can be created by introducinga targeting vector of the invention into a population of cells underconditions suitable for homologous recombination between the DNAencoding the tTA and the second target DNA molecule and selecting a cellin which the DNA encoding the tTA has integrated at a predeterminedlocation within the second target DNA molecule. The host cell can be amammalian cell (e.g., a human cell). Alternatively, the host cell can bea yeast, fungal or insect cell (e.g., the tTA-encoding DNA can beintegrated into a baculovirus gene within an insect cell). A preferredhost cell type for homologous recombination is an embryonic stem cell,which can then be used to create a non-human animal carrying tTA-codingsequences integrated at a predetermined location in a chromosome of theanimal. A host cell can further contain a gene of interest operablylinked to a tTA-responsive transcriptional promoter. The gene ofinterest operably linked to the tTA-responsive promoter can beintegrated into DNA of the host cell either randomly (e.g., byintroduction of an exogenous gene) or at a predetermined location (e.g.,by targeting an endogenous gene for homologous recombination). The genelinked to the tTA-responsive promoter can be introduced into the hostcell independently from the DNA encoding the tTA, or alternatively, a“single hit” targeting vector of the invention can be used to integrateboth tTA-coding sequences and a tTA-responsive promoter into apredetermined location in DNA of the host cell. Expression of a gene ofinterest operably linked to a tTA-responsive promoter in a host cell ofthe invention can be inhibited by contacting the cell with tetracyclineor a tetracycline analogue.

[0012] Another aspect of the invention relates to non-human transgenicanimals having a transgene comprising a polynucleotide sequence encodinga tetracycline-controllable transactivator (tTA) of the invention orhaving a transgene encoding a gene of interest operably linked to atTA-responsive promoter. Double transgenic animals having bothtransgenes (i.e., a tTA-coding transgene and a gene of interest linkedto a tTA-responsive promoter) are also encompassed by the invention. Inone embodiment, the transgenic animal is a mouse. In other embodiments,the transgenic animal is a cow, a goat, a sheep and a pig. Transgenicanimals of the invention can be made, for example, by introducing a DNAmolecule encoding the tTA or the gene of interest operably linked to atTA responsive promoter into a fertilized oocyte, implanting thefertilized oocyte in a pseudopregnant foster mother, and allowing thefertilized oocyte to develop into the non-human transgenic animal tothereby produce the non-human transgenic animal. Double transgenicanimals can be created by appropriate mating of single transgenicanimals. Expression of a gene of interest operably linked to a tTAresponsive promoter in a double transgenic animal of the invention canbe inhibited by administering tetracycline or a tetracycline analogue tothe animal.

[0013] Another aspect of the invention relates to non-human transgenicanimals having a transgene encoding a tTA of the invention, wherein thetransgene is integrated by homologous recombination at a predeterminedlocation within a chromosome within cells of the animal (also referredto herein as a homologous recombinant animal). The homologousrecombinant animal can also have a second transgene encoding a gene ofinterest operably linked to a tTA-responsive promoter. The secondtransgene can be introduced randomly or, alternatively, at apredetermined location within a chromosome (e.g., by homologousrecombination. For example, a single hit vector of the invention can beused to create a homologous recombinant animal in which expression ofthe tTA is controlled by 5′ regulatory elements of a gene of interestand expression of the gene of interest is controlled by thetTA-responsive promoter (such that in the absence of Tc, expression ofthe gene is stimulated by tTA binding to the tTA responsive promoter).

[0014] A non-human transgenic animal of the invention having tTA-codingsequences integrated at a predetermined location within chromosomal DNAof cells of the animal can be created by introducing a targeting vectorof the invention into a population of embryonic stem cells underconditions suitable for homologous recombination between the DNAencoding the tTA and chromosomal DNA within the cell, selecting anembryonic stem cell in which DNA encoding the tTA has integrated at apredetermined location within the chromosomal DNA of the cell,implanting the embryonic stem cell into a blastocyst, implanting theblastocyst into a pseudopregnant foster mother and allowing theblastocyst to develop into the non-human transgenic animal to therebyproduce the non-human transgenic animal.

[0015] Another aspect of the invention relates to a process forproducing and isolating a gene product (e.g., protein) encoded by a geneof interest operably linked to a tTA-responsive transcriptional promoterin a host cell of the invention carrying tTA-coding sequences. In theprocess, a host cell is first grown in a culture medium in the presenceof tetracycline or a tetracycline analogue (under these conditions,expression of the gene of interest is not stimulated). Next, theconcentration of tetracycline or the tetracycline analogue in theculture medium is reduced to stimulate transcription of the gene ofinterest. The cells are further cultured until a desired amount of thegene product encoded by the gene of interest is produced by the cells.Finally, the gene product is isolated from harvested cells or from theculture medium. Preferred cells for use in the process include yeast orfungal cells.

[0016] Kits containing the components of the regulatory system of theinvention described herein are also within the scope of the invention.

[0017] Various additional features, components and aspects of thisinvention are described in further detail below.

BRIEF DESCRIPTION OF THE FIGURES

[0018]FIG. 1 (panels a and b). Schematic representation of tetR-VP 16fusion proteins (tTAs), encoded by plasmids pUHD 15-1 and pUHD 151-1,and a tTA-dependent transcription unit, encoded by plasmid pUHC13-3.

[0019] Panel a: Diagrammatic representation of two tTA proteins. In bothfusion proteins, tTA and tTAS, the original 207-amino-acid sequence oftetR is conserved. Two versions of VP 16 sequences encoding theactivation domain were fused in frame to the 3′ end of the tetR gene,resulting in tTA and tTAs. The bold letters indicate the original aminoacids at the N terminal end, the junction, and the C-terminal end of thefusion proteins; the other letters designate amino acids introduced dueto sequence constraints of the particular system. The numbers delineateamino acid positions within tetR (Hillen and Wissman in Protein-NucleicAcid Interaction, Topics in Molecular and Structural Biology, Saengerand Heinemann (eds.), Vol 10, pp. 143-162 (1989)) or VP16 (Treizenberget al., Genes Dev. 2:718-729 (1988)), respectively.

[0020] Panel b: The tTA-dependent transcriptional unit consists of thesimian virus 40 (SV40) poly(A) site (An), the luciferase gene (luc), theP_(hCMV)*-1 or P_(hCMV)*-2. The two promoters encompass the sequencebetween +75 and −53 of the P_(hCMV)*-2 with one base-pair exchange at−31, which creates a Stu I cleavage site. The Xho I site introduced at−53 by PCR was utilized to insert the heptamerized tetO sequence. Thisheptameric sequence is flanked at one side by an 18-nucleotidepolylinker, which allows the insertion of the operators in bothorientations as Sal 1/Xhol fragments. The position of the central G/Cbase pair of the promoter proximal operator to position +1 is −95 forP_(hCMV)*-I (upper construct) and −76 for P_(hCMV)*-2 (lower construct).The plasmids that contain the four constructs are indicated on the farright.

[0021]FIG. 2 (panels a and b). Western blots showing the identificationand characterization of tTA produced in HeLa cells. HeLa cells grown to40% confluency were transiently transfected with pUHD15-1 by the calciumphosphate method. Nuclear and cytoplasmic extracts were prepared after36 hr.

[0022] Panel a: Western blot analysis of electrophoretically separatedextracts (6% acrylamide/0.1% SDS gels) with tetR-specific antibodiesreveals a protein of about 37 kDa (tTA) in cytoplasmic (C) and nuclear(N) extracts in pUHD 15-1 transfected cells (+) that is not present inmock-transfected cells (−).

[0023] Panel b: Mobility change of tetO DNA by tTA binding from HeLacell nuclear extracts. Radioactively labeled tetO DNA was mixed withextracts from mock-transfected (lanes 2 and 3) and pUHD15-1-transfected(lanes 4 and 5) HeLa cells in the absence (lanes 2 and 4) and presence(lanes 3 and 5) of 1 μg of tetracycline per ml (added 2 min prior to theaddition of the operator). Lane 1 contains labeled operator DNA only.

[0024]FIG. 3 (panels a and b). Graphs showing the dependence of tTAfunction on tetracycline.

[0025] Panel a: Dependence of luciferase (luc.) activity on tetracyclineconcentration. HeLa cell clones X1 (dashed line) and T12 previouslygrown in tetracycline-free medium were seeded with a density of 5000cells per 35 mm dish and incubated at the tetracycline concentrationsindicated. After reaching ˜90% confluency, cells were harvested andassayed for luciferase activity. Data given are the means ±SD of threeindependent experiments.

[0026] Panel b: Kinetics of tetracycline action. X1 cells were grown in100 mm dishes to 80% confluency in the absence or presence (0.1 μg/ml)of tetracycline. At time 0, cells were washed with phosphate-bufferedsaline and split into smaller culture dishes ({fraction (1/20)}th of theinitial cultures per 35 mm dish). Half of the cultures remained intetracycline-free medium (▪) and the other half were incubated in thepresence of tetracycline (1 μg/ml; □). The X1 culture grown intetracycline-containing medium was split in the same manner; one halfwas further incubated in the presence of tetracycline (), whereas theother half was transferred to tetracycline-free medium (∘). At the timesindicated, aliquots were harvested and examined for luciferase activity.The slight increase in luciferase activity monitored at 4 hr in theculture containing tetracycline () is reproducible and reflectsluciferase induction during the washing step.

[0027]FIG. 4. [SEQ ID NO: 1] The polynucleotide sequence coding for tTAtransactivator.

[0028]FIG. 5. [SEQ ID NO: 3]The polynucleotide sequence coding for tTAStransactivator.

[0029]FIG. 6. [SEQ ID NO: 5] The polynucleotide sequence of P_(hCMV)*-1.The nucleotide sequence shown encompasses the tet operator sequences(italics) and the hCMV minimal promoter, of which position −53, the TATAbox and position +75 (relative to the transcription start site) areunderlined.

[0030]FIG. 7. [SEQ ID NO: 6] The polynucleotide sequence of P_(hCMV)*-2. The nucleotide sequence shown encompasses the tet operatorsequences (italics) and the hCMV minimal promoter, of which position−53, the TATA box and position +75 (relative to the transcription startsite) are underlined.

[0031]FIG. 8. [SEQ ID NO: 7] The polynucleotide sequence of PTk*-1. Thenucleotide sequence shown encompasses the tet operator sequences(italics) and the HSV-Tk minimal promoter, of which position −81, theTATA box and position +7 (relative to the transcription start site) areunderlined.

[0032]FIG. 9A-9C. [SEQ ID NO: 8] The polynucleotide sequence of the cDNAcoding for the rabbit progesterone receptor under control of P_(hCMV)*-1

[0033]FIG. 10A-B. [SEQ ID NO: 9] The polynucleotide sequence of the cDNAcoding for the rabbit progesterone receptor under control Of P_(hCMV)*-1

[0034]FIG. 11. A schematic representation of Conditional Knock OutStrategy 1 in which “E.G. 5′” represents flanking nucleotide sequencefrom 5′ of the coding sequence for an Endogenous Gene; “E.G. 3′”represents flanking nucleotide sequence from 3′ of the coding sequencefor an Endogenous Gene; and “tTARE” represents a tTA responsive elementinserted just upstream of a copy of the endogenous gene of interest. (Inother embodiments the gene linked to the tTA is a heterologous gene.)

[0035]FIG. 12. A schematic representation of Conditional Knock OutStrategy 2 in which “tTARE” is a tTA responsive promoter element: “E.G”.is an endogenous gene; “E.G. 5′” represents flanking nucleotide sequencefrom 5′ of the coding sequence for an Endogenous Gene; “E.G. 3”represents flanking nucleotide sequence from 3′ of the coding sequencefor an Endogenous Gene; and “TK” is a thymidine kinase gene.

[0036]FIG. 13.A-B A schematic representation of Conditional Knock OutStrategy 3 depicting vector designs in which abbreviations are asdefined above, Neo^(r) is a neomycin resistance gene and pPGK isphosphoglycerate kinase sequence.

[0037]FIG. 14. A graphic representation of the doxycycline dependentluciferase activity in double transgenic mice carrying P_(hCMV)-tTA andP_(hCMV)*-1-luc transgenes. Light bars show tTA-activated luciferaselevels in different tissues from 2 individual mice. Dark bars showluciferase levels in different tissues from 2 individual mice thatreceived doxycycline in the drinking water (200 mg/ml, 5% sucrose) for 7days. Spotted bars (controls) represent the average luciferasebackground activity from 5 individuals from line L7, carrying only theP_(hCMV)*-1-luc transgene.

DETAILED DESCRIPTION OF THE INVENTION

[0038] Definitions

[0039] The description that follows makes use of a number of terms usedin recombinant DNA technology. In order to provide a clear andconsistent understanding of the specification and claims, including thescope to be given such terms, the following definitions are provided.

[0040] Cloning Vector. A plasmid or phage DNA or other DNA sequencewhich is able to replicate autonomously in a host cell, and which ischaracterized by one or a small number of restriction endonucleaserecognition sites at which such DNA sequences may be cut in adeterminable fashion without loss of an essential biological function ofthe vector, and into which a DNA fragment may be spliced in order tobring about its replication and cloning. The cloning vector may furthercontain a marker suitable for use in the identification of cellstransformed with the cloning vector.

[0041] Expression Vector, A vector similar to a cloning vector but whichis capable of enhancing the expression of a gene which has been clonedinto it, after transformation into a host. The cloned gene is usuallyplaced under the control of (i.e., operably linked to) certain controlsequences such as promoter sequences. Promoter sequences may be eitherconstitutive or inducible.

[0042] Eucaryotic Host Cell. According to the invention, a eucaryotichost cell may be any such cell which include, but are not limited to,yeast cells, plant cells, fungal cells, insect cells, e.g. Schneider andsF9 cells, mammalian cells, e.g. HeLa cells (human), NIH3T3 (murine),RK13 (rabbit) cells, embryonic stem cell lines, e.g, D3 and J1, and celltypes such as hematopoietic stem cells, myoblasts, hepatocytes,lymphocytes, airway epithelium and skin epithelium.

[0043] Recombinant Eucaryotic Host. According to the invention, arecombinant eucaryotic host may be any eucaryotic cell which containsthe polynucleotide molecules of the present invention on an expressionvector or cloning vector. This term is also meant to include thoseeucaryotic cells that have been genetically engineered to contain thedesired polynucleotide molecules in the chromosome, genome or episome ofthat organism. Thus, the recombinant eucaryotic host cells are capableof stably or transiently expressing the proteins.

[0044] Recombinant vector. Any cloning vector or expression vector whichcontains the polynucleotide molecules of the invention.

[0045] Host. Any prokaryotic or eucaryotic cell that is the recipient ofa repiicable vector. A “host” as the term is used herein, also includesprokaryotic or eucaryotic cells that can be genetically engineered bywell known techniques to contain desired gene(s) on its chromosome orgenome. For examples of such hosts, see Sambrook et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989).

[0046] Promoter. A DNA sequence generally described as the 5′ region ofa gene, located proximal to the start codon. The transcription of anadjacent gene(s) is initiated at the promoter region. If a promoter isan inducible promoter, then the rate of transcription increases inresponse to an inducing agent. In contrast, the rate of transcription isnot regulated by an inducing agent if the promoter is a constitutivepromoter.

[0047] Minimal Promoter. A partial promotor sequence which defines thetranscription start site but which by itself is not capable, if at all,of initiating transcription efficiently. The activity of such minimalpromoters depend on the bindiny of activators such as atetracycline-controlled transactivator to operably linked binding sites.

[0048] Gene. A DNA sequence that contains information needed forexpressing a polypeptide or protein.

[0049] Structural Gene. A DNA sequence that is transcribed intomessenger RNA (mRNA) that is then translated into a sequence of aminoacids characteristic of a specific polypeptide.

[0050] Polynucleotide molecules. A polynucleotide molecule may be apolydeoxyribonucleic acid molecule (DNA) or a polyribonucleic acidmolecule (RNA).

[0051] Complementary DNA (cDNA). A “complementary DNA” or “cDNA” geneincludes recombinant genes synthesized by reverse transcription of mRNAand from which intervening sequences (introns) have been removed.

[0052] Expression. “Expression” is the process by which a polypeptide isproduced from a structural gene. The process involves transcription ofthe gene into mRNA and the translation of such mRNA into polypeptide(s).

[0053] Fragment. A “fragment’ of a molecule is meant to refer to anypolypeptide subset of that molecule.

[0054] Tet repressor. A “tet repressor” refers to a prokaryotic proteinwhich binds to a tet operator sequence in the absence but not thepresence of tetracycline. The term “tet repressor” is intended toinclude repressors of different class types, e.g., class A, B, C, D or Etet repressors.

[0055] Tetracycline Analogue. A “tetracycline analogue” is any one of anumber of compounds that are closely related to tetracycline (Tc) andwhich bind to the tet repressor with a Ka of at least about 10⁶ M⁻¹.Preferably, the tetracycline analogue binds with an affinity of about10⁹ M⁻¹ or greater, e.g. 10⁹M⁻¹. Examples of such tetracycline analoguesinclude, but are not limited to those disclosed by Hlavka and Boothe,“The Tetracyclines,” in Handbook of Experimental Pharmacology 78, R. K.Blackwood et al. (eds.), Springer Verlag, Berlin-New York, 1985; L. A.Mitscher “The Chemistry of the Tetracycline Antibiotics, MedicinalResearch 9, Dekker, N.Y., 1978; Noyee Development Corporation,“Tetracycline Manufacturing Processes,” Chemical Process Reviews, ParkRidge, N.J., 2 volumes, 1969; R. C. Evans, “The Technology of theTetracyclines,” Biochemical Reference Series 1, Quadrangle Press, NewYork, 1968; and H. F. Dowling, “Tetracycline,” Antibiotics Monographs,no. 3, Medical Encyclopedia, New York, 1955; the contents of each ofwhich are fully incorporated by reference herein. Examples oftetracycline analogues include anhydrotetracycline, doxycycline,chlorotetracycline, epioxytetracycline, cyanotetracycline and the like.Certain Tc analogues, such as anhydrotetracycline andepioxytetracycline, have reduced antibiotic activity compared to Tc.

[0056] Transgenic Animal. A transgenic animal is an animal having cellsthat contain a transgene, wherein the transgene was introduced into theanimal or an ancestor of the animal at a prenatal, e.g., an embryonic,stage. A transgene is a DNA which is integrated into the genome of acell from which a transgenic animal develops and which remains in thegenome of the mature animal, thereby directing the expression of anencoded gene product in one or more cell types or tissues of thetransgenic animal. Non-human animals into which transgenes can beintroduced by techniques known in the art include mice, goats, sheep,pigs, cows and other domestic farm animals.

[0057] A transgenic animal can be created, for example, by introducing anucleic acid encoding a protein of interest (typically linked toappropriate regulatory elements, such as a constitutive ortissue-specific enhancer) into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, and allowing the oocyte to develop in apseudopregnant female foster animal. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Methods forgenerating transgenic animals, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009 and Hogan, B. et al., (1986) ALaboratory Manual, Cold Spring Harbor, N.Y., Cold Spring HarborLaboratory. A transgenic founder animal can be used to breed additionalanimals carrying the transgene. A transgenic animal carrying onetransgene can further be bred to another transgenic animal carrying asecond transgenes to create a so-called “double transgenic” animalcarrying two transgenes.

[0058] Homologous Recombinant Animal. The term “homologous recombinantanimal” as is used herein is intended to describe an animal containing agene which has been modified by homologous recombination between thegene and a DNA molecule introduced into an embryonic cell of the animal,or ancestor thereof. Thus, a homologous recombinant animal is a type oftransgenic animal in which the transgene is introduced into apredetermined chromosomal location in the genome of the animal byhomologous recombination.

[0059] To create such a homologous recombinant animal, a vector isprepared which contains DNA of interest (e.g., encoding a tTA of theinvention) flanked at its 5′ and 3′ ends by additional nucleic acid of aeucaryotic gene of interest at which homologous recombination is tooccur. The flanking nucleic acid is of sufficient length for successfulhomologous recombination with the eucaryotic gene. Typically, severalkilobases of flanking DNA (both at the 5′ and 3′ ends) are included inthe vector (see e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell51:503 for a description of homologous recombination vectors). Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected (see e.g., Li, E. et al.(1992) Cell 69:915). The selected cells are then injected into ablastocyst of an animal (e.g., a mouse) to form aggregation chimeras(see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: APractical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp.113-152). A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term.Progeny harbouring the homologously recombined DNA in their germ cellscan be used to breed animals in which all cells of the animal containthe homologously recombined DNA by so-called “germline transmission”.Animals carrying the recombined gene can be bred to homozygosity and/orbred with other animals carrying other transgenes.

[0060] Recombinant expression of proteins is commonly done usingconstitutive promoters like human CMV (Boshart, M. et al. 1985, CellVol. 41, 521-530) or the adenovirus major late promoter or SV40 earlypromoter as described below (see also, e.g., Kaufman, R. J. 1990 MethEnzymol. Vol. 185: 537-566 and Benoist C. et al. (1981) NatureVol.290:304 ff).

[0061] However, in the case of proteins such as certain proteases,cytotoxic or cytostatic proteins that interfere with the cell membranesor proteins like certain receptors, whose normal biological functiontriggers a response to the host cell environment (media components,temperature etc.) that is detrimental to the host cell, expression ofthe proteins may negatively effect the physiology of the host cell. Inother cases overexpression of a desired gene may simply be unduly taxingfor the producing cells. In such cases it is desirable to inhibit theexpression of the desired gene until an optimal cell density has beenachieved, and only then, after an optimal period of cell culture invitro or cell growth and development in vivo (determined empirically),induce gene expression in the cells to produce sufficient quantities ofthe protein. While a number of systems have been proposed and tried (asgenerally reviewed by Yarranton, G. T. 1992 Current Opinion inBiotechnology Vol. 3:506-511) many such systems do not allow for tightrepression and subsequent complete activation. Others employ impracticalactivation steps that are not expected to be useful in large scalefermentation or in whole animals. The current invention however fulfillsall these criteria in eucaryotic expression systems using atranscriptional switch based on procaryotic control elements.

[0062] Aspects of the tightly regulatable genetic switch used in thisinvention for controlling gene transcription are described in Gossen &Bujard, 1992, Proc. Natl. Acad. Sci. USA 89:55475551 and in U.S. patentapplication Ser. No. 08/076,726, entitled “Tight Control of GeneExpression in Eucaryotic Cells by Tetracycline-responsive Promoters”filed Jun. 14, 1993, the full contents of both of which are incorporatedherein by reference.

[0063] The genetic switch employed in this invention comprises twocomponents: (i) a polynucleotide (e.g. DNA) moiety encoding atetracycline-controllable transcriptional activator (also referred toherein as a “transactivator” or tTA) and (ii) a gene of interestoperably linked to, i.e., under the transcriptional control of, apromoter responsive to the transcriptional activator.

[0064] The tetracycline-controllable transactivator (tTA) is composed ofa procaryotic tet repressor (tetR) (also referred to as the firstpolypeptide) operably linked to a polypeptide which directly orindirectly activates transcription in eucaryotic cells (also referred toas the second polypeptide). Typically, nucleotide sequences encoding thefirst and second polypeptides are ligated to each other in-frame tocreate a chimeric gene encoding a fusion protein, although the first andsecond polypeptides can be operably linked by other means that preservethe function of each polypeptide (e.g., chemically crosslinked). In oneembodiment, the second polypeptide is a transcriptional activatingprotein such as the acidic transactivating domain of virion protein 16(VP16) of herpes simplex virus (HSV) as in plasmids pUHD15-1 orpUHD151-1 (see FIG. 11). It should be appreciated that othertransactivators, including acidic, proline- or serine/threonine- orglutamnine-rich transactivating moieties as described below, may besubstituted for the VP16 transactivator in the tetracycline-controllablefusion transactivator. In this embodiment, the second polypeptide of thefusion protein is capable of directly activating transcription.

[0065] In another embodiment, the second polypeptide of the tTA fusionprotein indirectly activates transcription by recruiting atranscriptional activator to interact with the tetR fusion protein. Forexample, tetR can be fused to a polypeptide domain (e.g., a dimerizationdomain) capable of mediating a protein-protein with a transcriptionalactivator protein, such as an endogenous activator present in a hostcell. It has been demonstrated that functional associations between DNAbinding domains and transactivation domains need not be covalent (seee.g., Fields and Song (1989) Nature 340:245-247; Chien et al. (1991)Proc. Natl. Acad. Sci. USA 88:9578-9582; Gyuris et al. (1993) Cell75:791-803; and Zervos, A. S. (1993) Cell 72:223-232). Accordingly, thesecond polypeptide of the tTA fusion protein may not directly activatetranscription but rather may form a stable interaction with anendogenous polypeptide bearing a compatible protein-protein interactiondomain and transactivation domain. Examples of suitable interaction (ordimerization) domains include leucine zippers (Landschulz et al. (1989)Science 243:1681-1688), helix-loop-helix domains (Murre, C. et al.(1989) Cell 58:537-544) and zinc finger domains (Frankel, A. D. et al.(1988) Science 240:70-73). Interaction of a dimerization domain presentin the tTA fusion protein with an endogeneous nuclear factor results inrecruitment of the transactivation domain of the nuclear factor to thetTA, and thereby to a tet operator sequence to which the tTA is bound.

[0066] A variation of this approach is to construct a fusion of the tetRDNA binding sequence to the non-DNA binding amino acid sequences of theTATA binding protein (TBP) (TBP is described in Kao, C. C. et al. (1990)Science 248:1646-1650). The DNA binding form of TBP is part of a proteincomplex designated TFIID. The function of TBP in the complex is torecruit other protein components of the TFIID complex to position nearthe transcription initiation site of eucaryotic genes containing a TATAbox (i.e., TBP binding site). When bound to the TATA box, the TFIIDcomplex subsequently mediates the sequential recruitment of othermembers of the basic transcriptional initiation complex, resulting ininitiation of transcription (described in more detail in Buratowski, S.et al. (1989) Cell 56:549-561). Accordingly, when fused to tetR DNAbinding sequences, TBP may recruit other members of the basictranscription initiation complex to DNA sequences containing a tetoperator(s). Furthermore, by substituting a TATA sequence present in aeukaryotic gene of interest with a tet operator(s), the tetR/TBP fusionprotein can be targeted to this site in a manner dependent on thepresence or absence of Tc (or analogue thereof), resulting inTc-dependent initiation of transcription. Since, in this approach, thegene of interest to be regulated by the tTA (i.e., tetR/TBP fusionprotein) lacks a functional TATA element, the basal level of expressionof the gene in the presence of Tc (or analogue) is expected to be verylow. However, upon removal of Tc (or analogue), transcription initiationis restored via binding of tetR/TBP to the tet operator(s) andrecruitment of other components of the transcription initiation complex.

[0067] The tTA may be expressed in a desired host cell using otherwiseconventional methods and materials by transfecting or transforming thehost cell with the tTA-encoding DNA operably linked to a conventionalpromoter (such as are mentioned elsewhere herein), e.g. for constitutiveexpression.

[0068] The second component of the genetic switch is the tTA-responsivetranscriptional promoter to which the gene of interest is operablylinked. The promoter may be a minimal promoter comprising, for example,a portion of the cytomegalovirus (CMV) IE promoter, operably linked toat least one tet operator sequence, derived for example from thetetracycline resistance operon encoded in Tn10 of E coli (Hillen &Wissmann, “Topics in Molecular and Structural biology” inProtein-Nucleic Acid Interaction, Saeger & Heinemannn eds., Macmillan,London, 1989, Vol.10, pp.143-162), to serve as target sequences for atTA. Other suitable minimal promoters include P_(hCMV)*-1, P_(hCMV)*-2,and PtK*-1, described herein, or other minimal promoters derived frompromoter elements typically used in the cell line employed as describedin the references throughout this application.

[0069] Minimal promoter elements particularly useful for a given cellline may be selected from a series of deletion mutants of the originalpromoter nucleotide sequence, based on the ability of a given member ofthe series (for instance, placed as a Xhol/Sac11 fragment into thecorresponding restriction sites of plasmid pUHC13-3) to be activated intransient transfection experiments using a cell line stably expressingthe tetR-VP16 fusion protein; as will be appreciated a cell line stablyexpressing any other fusion of tetR with a protein domain capable ofactivating transcription (see below) can be used. As will also beappreciated plasmid pUHC13-3 may be modified for the specificapplication by replacing genetic elements like poly-adenylation sites orsplice sites with those functioning in the cell line in question.Specific details may be found in the references throughout thisapplication or references cited therein, the full contents of which areincorporated herein by reference. A second criterion for the selectionof the optimal minimal promoter is the degree of repression in thepresence of tetracycline (see below). Typically the deletion mutant withthe highest activation factor as described below is chosen.

[0070] Promoter deletion mutants may be prepared as generally describedby Rosen, C. et al (1985) Cell Vol.41, 813-823 or Nelson C. et al.(1986) Nature Vol.322, 557-562. Other methods, including methods usefulin the preparation of stable tetR-VP16 cell lines, are essentially asdescribed in “Current Protocols in Molecular Biology” Ausubel, F. M. etal (eds.) 1989 Vol. 1 and 2 and all supplements to date GreenePublishing Associates and Wiley-Interscience, John Wiley & Sons, NewYork, the full contents of which are incorporated herein by reference,or as described in the other references cited throughout thisapplication.

[0071] The presence of tet operator element(s) renders such recombinantpromoter moieties responsive to the tTA of the invention. In HeLa cellsconstitutively expressing the TetR-VP16 tTA, high levels of luciferaseexpression have been achieved under the control of such a modified CMVpromoter sequence. The incorporation of the tetR domain within the tTArenders this expression system sensitive to the presence oftetracycline. The binding of tetracycline to the tetR domain of the tTAprevents the tTA from exerting its transactivating effects. Depending onthe concentration of tetracycline in the culture medium (0-1 μg/ml), theluciferase activity can be regulated up to five orders of magnitude inthe previously mentioned example. This system provides both a reversibleon/off switch and a differential control—as desired—for regulating geneexpression in eucaryotic hosts. It should be appreciated thattetracycline analogs which are capable of specific functionalinteraction with tetR may be used in place of tetracycline,

[0072] A eucaryotic production cell line of this invention is preparedaccording to the design described above. Assembly of the components andincorporation thereof into a eucaryotic host cell are conducted byotherwise conventional methods such as are described generally byKriegler, M. (editor), 1990, Gene Transfer and Expression, A LaboratoryManual (Stockton Press). Care should to be taken to select forintegration of the gene of interest into a chromosomal site thatexhibits sufficiently low basal expression when, or to the extent,desired (see e.g. Table 1). The recombinant host cell obtained is grownin the presence of tetracycline or tetracycline analogues until anoptimal density that has been determined empirically to allow forsubsequent induction of gene expression. After the desired cell densityhas been reached gene expression is induced by dilution and/or removalof the tetracycline or analog thereof. The culture is then continuouslygrown until a optimal expression level has been reached. The recombinantprotein is then harvested according to standard procedures.

[0073] The use of eucaryotic cells as host cells for expression ofrecombinant proteins is generally reviewed in M. Kriegler 1990 “GeneTransfer and Expression, A Laboratory Manual”. Stockton Press.,incorporated herein as reference. While CHO^(dhfr-) cells (Urlaub, G.and Chasin (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220), 293 cells(Graham, F. L. et al. (1977) J. Gen. Virol. 36: pp. 59) or myeloma cellslike SP2 or NSO (Galfre, G. and Milstein, C. (1981) Meth Enzymol. 73(B): 3-46) are commonly used it should be clear to those of ordinaryskill in the art, that any eucaryotic cell line can be used in thepractice of the subject invention, so long as the cell line is notincompatible with the protein to be expressed, the selection systemchosen or the fermentation system employed. This invention is broadlyapplicable and encompasses non-mammalian eucaryotic cells as well,including insect (e.g. Sp. frugiperda), yeast (e.g. S. cerevisiae, S.pombe, H. polymorpha) and fungal cells, containing and capable ofexpressing the two components of the foregoing genetic switch.

[0074] The eucaryotic host cells used for regulated expression in thisinvention may thus be yeast cells including, but not limited toSaccharomyces cerevisiae, Pichia pastoris, Kluyveromyces lactis andHansenula polymorpha, as generally reviewed by Fleer, R. (1992), CurrentOpinion in Biotechnology Vol. 3, No. 5: p. 486-496, the full contentsthereof and of which the references cited therein are incorporatedherein by reference.

[0075] In other embodiments the eucaryotic cells used for regulatedexpression are insect cells carrying in their chromosomes theheterologous DNA moiety encoding a transactivator fusion protein (tTA)comprising a tetracycline repressor and a protein capable of activatingtranscription in the host cell. A second recombinant DNA moiety encodingthe gene of interest operably linked to a promoter responsive to thetranscriptional activator is carried on the baculovirus genome. Suitablegeneral methods which may be used in the practice of these aspects ofthe invention are reviewed by O'Reilly et al. (1992)“Baculovirusexpression vectors, A Laboratory Manual” Stockton Press, the fullcontents of which are incorporated herein by reference.

[0076] While the gene of interest may be a heterologous gene, i.e. nototherwise present in the parental host cell genome, an important aspectof this invention relates to the regulation of an endogenous gene ofinterest. In such cases the host cell is genetically engineered toinsert into the host cell genome the tTA-responsive promoter such thatthe desired endogenous gene is under the transcriptional control of thetTA-responsive promoter. This may be accomplished for example by linkinga copy of the endogenous gene to the tTA-responsive promoter andtransfecting or transforming the host cell with the recombinantconstruct. In one approach, the construct is introduced by homologousrecombination into the loci of the endogenous gene. Briefly, the tTAresponsive promoter is flanked on the 5′ side by sufficient DNAsequences from the upstream region but excluding the actual promoterregion of the endogenous gene and on the 3′ end by sequencesrepresenting the coding region of the endogenous gene. The extent of DNAsequence homology necessary for homologous recombination is discussedbelow.

[0077] In other approaches that construct is inserted at another geneticlocus, either predetermined or at random. In any case, the eucaryoticcell is also transformed or transfected with the DNA constructpermitting expression of the tTA. Alternatively, the DNA constructencoding the tTA may itself be inserted at the locus of the endogenousgene of interest and the DNA moiety encoding the gene of interestoperably linked to a tTA-responsive promoter may be introduced elsewherein the genome. In that embodiment, the tTA vector contains thetTA-encoding DNA moiety flanked by DNA sequence of the locus of theendogenous gene permitting homologous recombination of the constructinto that locus.

[0078] The use of flanking DNA sequence to permit homologousrecombination into a desired genetic locus is known in the art. Atpresent it is preferred that up to several kilobases or more of flankingDNA corresponding to the chromosomal insertion site be present in thevector on both sides of the tTA-encoding sequence (or any other sequenceof this invention to be inserted into a chromosomal location byhomologous recombination) to assure precise replacement of chromosomalsequences with the exogenous DNA. See e.g. Deng et al, 1993, Mol. Cell.Biol 13(4):2134-40; Deng et al, 1992, Mol Cell Biol 12(8):3365-71; andThomas et al, 1992, Mol Cell Biol 12(7):2919-23. It should also be notedthat the eucaryotic cell of this invention may contain multiple copiesof the gene of interest, e.g. by conventional genetic amplification,each operably linked to the tTA-responsive promoter.

[0079] It should be clear from the preceding that to achieve the goalsof introducing the DNA moiety encoding the tTA into the host cell genomeand of introducing the tTA-responsive promoter construct in operablelinkage to the desired gene, vectors based on the following principlesare required. First, to introduce the tTA-encoding construct into thegenome of the host cell such that its expression will follow theregulated pattern of expression observed in the unmodified host cell forthe gene of interest, it is necessary to introduce the tTA-encodingconstruct such that its expression is made subject to the transcriptioncontrol elements associated with the gene of interest. One way to do sois to introduce the tTA-encoding construct by homologous recombinationinto the genetic locus of the gene of interest. A vector for suchintroduction comprises the DNA sequence encoding the tTA flanked bysufficient DNA sequence from the locus of the gene of interest in thehost genome to permit the desired homologous recombination event inwhich the tTA and flanking DNA is effectively swapped for the flankingDNA copy and the DNA included therebetween within the host cell genome.As will be appreciated an expression construct containing a tTAresponsive promoter operably linked to the DNA sequence of theendogenous gene can be integrated at random sites without the help offlanking homologous sequences as described in references throughout thisapplication. Alternatively, to insert a DNA sequence comprising atTA-responsive promoter or tetO control element(s) upstream of a desiredgene, a construct is assembled in which the DNA comprising thetTA-responsive promoter is ligated upstream of a copy of the desiredgene between DNA sequences flanking the desired insertion site in thehost genome. In either event the tTA construct can be introduced asmentioned previously.

[0080] Using the foregoing genetic constructs and engineered eucaryoticcells, this invention further provides a method for regulating theexpression of a gene of interest. In one aspect of this methodeucaryotic host cells engineered as described above are cultured underotherwise conventional conditions suitable for cell growth andproliferation, but in a culture medium containing a substance capable ofbinding to the tetracycline repressor moiety and of blocking orinhibiting transcriptional activation. Tetracycline is the archetypicalsuch substance. However, tetracycline analogs which bind to tetR to forma complex which is not transcriptionally activating may of course besubstituted for tetracycline. The precise concentration of tetracyclineor other such substance will depend on the substance's affinity for thetetR domain and/or the substance's specific inhibitory activity, as wellas the cell density and copy number of the tTA and the desired level ofinhibition of gene expression.

[0081] Nonetheless, appropriate levels of inhibitory substance for thedesired level of inhibition are. readily determinable empiricallywithout undue effort.

[0082] Cell culture in accordance with the preceding method negativelyregulates, i.e. inhibits expression of the gene of interest, completelyor partially. Culturing of the cells thereafter in media with a lowerconcentration (relative to the initial concentration) of the tetRbinding substance permits gene expression to begin or to ensue at a nowhigher level. If an initial concentration of binding substance (e.g.tetracycline) is selected which is sufficient to inhibit genetranscription substantially completely (e.g. transcription is notobserved under conventional Northern blotting conditions), and in thefollowing phase of cell culture the binding substance is substantiallyremoved from the media, gene expression can be said to be regulated inan on/off manner. In some applications, intermediate levels ofexpression may be desired. To that end, concentrations of bindingsubstance may be selected based on empirical data to providepredetermined intermediate level(s) of gene transcription. It should beunderstood that removal of the binding substance from the media may beeffected by gradual, step-wise, continual or total replacement ofculture media containing the binding substance with culture medialacking the binding substance or simply containing reduced levels of thebinding substance.

[0083] Where the eucaryotic cells engineered in accordance with thisinvention are incorporated into the host organism, e.g. to create atransgenic organism, this invention provides a genetically engineerednon-human animal capable of regulatably expressing a gene of interest.Such animal, in the broad sense, comprises cells containing and capableof expressing a heterologous DNA moiety encoding a tTA as previouslydefined and a DNA moiety comprising an gene of interest under thetranscriptional control of a heterologous promoter responsive to thetranscriptional activator.

[0084] Thus, this invention further relates to non-human animals derivedby homologous recombination of one or more polynucleotide molecules ofthe invention into a specific target site within their genome, theoffspring of such animals, as well as to a method to prevent or promotethe expression of a targeted gene in a conditional manner.

[0085] This embodiment of the invention is able to solve a longstandingproblem in the field generally described as gene targeting or gene knockout (Capecchi. M. R. (1989) Science Vol 244, p. 1288-1292, Bradley, A.(1991) Current opinion in Biotechnology Vol. 2, p. 823-829) pertainingto genes whose mutations results in death of the homozygous embryos,e.g., as described for the knock out of the RB gene (Jacks, T. et al.(1992) Nature 359:295-300). If the genetic switch subject of the currentinvention is applied as described below, expression of an endogenousgene of interest operably linked to a tet operator sequence(s) can bestimulated by a tetracycline-controllable transactivator (tTA) of theinvention and the animal develops like a nonmutated wildtype animal.Then, at a particular stage of development, expression of theendogeneous gene of interest can be switched off by raising the level oftetracycline or a tetracycline analogue in the circulation and thetissues of the animal by feeding or injecting the tetracycline ortetracycline analog to the animal, thereby inhibiting the activity ofthe tTA and transcription of the gene of interst. This method isgenerally referred to herein as a “conditional knockout”.

[0086] As will be clear from the following, two principally differentapproaches have been devised to apply the genetic switch of thisinvention to the genome of the non-human animal in a way, that willallow for a temporally and spatially correct expression of theendogenous gene. In one approach, the two elements of the genetic switchare in separate locations in the chromosome and require two integrationsteps, another one achieves the desired result in one step.

[0087] In the first step of one embodiment of the invention non-humananimals are derived by homologous recombination of the DNA sequences ofthe tTA into a specific DNA site containing the nucleotide sequences ofan endogenous gene of interest in such a way that part or all of thecoding sequence of the endogenous gene is replaced with the tTA gene.This can be accomplished (see FIG. 11) in the following steps:

[0088] (1) assembling a chimeric gene in which the sequence of the first(i.e. tTA) polynucleotide molecule of the invention is flanked by DNAsequences from the gene of interest such that upon incorporation of thechimeric gene into the host genome, the DNA sequences that normallycontrol the expression of the target gene are fused to and controlexpression of the DNA sequences for the tTA.

[0089] (2) introducing this chimeric gene into an embryonic stem cellline from a species of interest and screening resultant candidateembryonic cell clones to identify and recover those cells in whichhomologous recombination has taken place at the locus of interest.

[0090] (3) introducing those recombinant cells so identified andrecovered into a blastocyst from the species of interest to yield achimeric embryo.

[0091] (4) implanting the chimeric embryo into the uteri ofpseudopregnant recipient mothers to facilitate development and birth ofa homologous recombinant offspring.

[0092] This process results in offspring whose genome contains the DNAsequence encoding the tTA inserted in place of the gene of interest suchthat the tTA DNA is expressed in a pattern similar or identical to thatof the gene of interest. These processes and their results arecollectively and commonly referred to as “gene knock-out”. Thesetechniques are well established and described in: Wood et al. Proc.Natl. Acad. Sci. 90:4582-4585, Simon et al. Nature Genetics 1:92-97 &Soriano et al. Cell 64:693-702 and references therein, the full contentof which are in their entirety incorporated herein by reference.

[0093] The second step in this embodiment of the invention relates tothe preparation of a second transgenic animal which contains in it'sgenome the gene of interest under transcriptional control of thetetracycline (Tc) responsive promoter element. This can be accomplishedusing the following method:

[0094] (1) A chimeric DNA sequence is prepared where a Tc responsivepromoter element, (comprising at least one tet operator and a minimalpromoter) is cloned 5′ of the DNA sequences encoding the endogenous geneof interest. One way to accomplish this is to replace the luciferasecoding sequence and all polyadenylation elements in the plasmidspUHC13-3 or pUHC13-4 with the DNA sequence containing the completegenomic coding sequence of the endogenous gene and sufficient 3′ noncoding sequence to allow for proper polyadenylation. As will beappreciated the DNA sequence encoding the endogenous gene can also becDNA (cloned as an example in such a way that it replaces exactly theluciferase gene in pUHC13-3 or pUHC13-4) or any combination of genomicDNA and cDNA designed to provide the complete coding sequence, anyregulatory elements that may reside in intron sequences or is notcontained in it's entirety in the cDNA and a polyadenylation signal orother elements typically associated with the endogenous gene. Generalcloning and DNA manipulation methods are described in references citedthroughout this application.

[0095] (2) The chimeric DNA sequence (called also “the chimerictransgene”) is injected into a fertilized egg which is implanted into apseudopregnant recipient mother and allowed to develop into an adultanimal. In particular, a few hundred DNA molecules are injected into thepro-nucleus of a fertilized one cell egg. The microinjected eggs arethen transferred into the oviducts of pseudopregnant foster mothers andallowed to develop. It has been reported by Brinster et al. (1986) Proc.Natl. Acad. Sci. USA Vol. 83:9065-9069, the full contents of which areincorporated by reference herein, that about 25% of mice which developwill inherit one or more copies of the microinjected DNA. A protocol forconstructing such transgenic animals (Brinster et al. Proc. Natl. Acad.Sci. 83:4432-4445, Crenshaw et al. Genes 3: Dev 9:959-972 and referencescited therein) is a well established technique as is the breeding ofrecombinant and hybrid animals.

[0096] Breeding of animals resulting from the first and the second stepof this embodiment of the invention produces offspring containing boththe replaced gene of interest and the chimeric transgene. In a preferredembodiment, animals heterozygous for the knockout of the endogenous generesulting from the first step of this embodiment of the invention (andinstead expressing a tTA gene) are used for breeding with animals thatare homozygous for the chimeric transgene resulting from the second stepof this embodiment of the invention. The resulting offspring areanalyzed by standard techniques, including tail-blot analysis describedin references throughout this application, and animals homozygous forboth traits are selected. Typically about 50% of the offspring shouldcarry both traits. In these animals, replacement of the coding sequencesof the gene of interest with those of the DNA sequences of the tTA issuch that the tTA is expressed in a temporal and spatial pattern similaror identical to that of the gene of interest and regulates in transexpression of the gene of interest now under transcriptional control of(i.e., operably linked to) the DNA sequences of the Tet operator andminimal promoter inserted at it's 5′ end.

[0097] As will be appreciated, the particular breeding strategy dependson the nature of the gene of interest. If the “knock out” of theendogenous gene with the tTA coding sequence is not lethal and theoverall plan is to create animals where the functions of the gene ofinterest in the adult can be studied in the “on” or “off” state, theanimals from the first step of this embodiment of the invention can bebred to homozygosity and then bred with the homozygous mice from thesecond step.

[0098] In this combination, the gene of interest is regulated by theaddition or substraction of tetracycline or its analogs from the food orwater supply of the animal as discussed below.

[0099] In another embodiment of the invention, embryonic stem (ES) celltechnology is used to prevent or promote expression of a gene interestin a conditional manner (FIG. 12). In the first step of this embodimentof the invention, a chimeric DNA sequence (commonly referred to as achimeric transgene) consisting of the DNA sequences of the tetoperator(s) and a suitable minimal promoter inserted 5′ of the DNAsequences encoding a gene of interest is introduced by stable,non-homologous recombination into random sites in the ES cell genome.Co-introduced with this chimeric construct is a selectable marker thatenables the selection of cell clones that have integrated DNA constructsfrom cells that have not. As will be appreciated, the feeder cellssupporting the growth of the ES cells have to express the sameresistance gene used for the selection step. As an example, if theselection marker chosen is the hygromycin resistance gene, the primaryfeeder layer cells used for the ES cell culture can be derived from ananimal transgenic for the hygromycin resistance gene prepared accordingto standard procedures for the preparation of transgenic animals, ascited throughout this application. ES cell clones are selected for lowbasal expression of the chimeric transgene using customary detectionmethods, such as evaluating the mRNA levels of the transgene asdescribed in “Current Protocols in Molecular Biology” Ausubel, F. M. etal (eds.) 1989 Vol. 1 and 2 and all supplements to date, GreenePublishing Associates and Wiley-Interscience, John Wiley & Sons, NewYork, the fall contents of which are incorporated herein by reference,or as described in the other references cited throughout thisapplication. Other methods to detect expression of the transgene mayinclude activity assays or assays designed to detect protein expression.Low basal expression of the transgene is determined relative tountransfected cells. Alternatively, low base 1 expression of the tetoperator-linked transgene can be evaluated in different tissues ofanimals derived from the embryonic stem cells. For example, ES cells canbe transfected with the transgene in culture, and the clones expanded,selected and injected into blastocysts to create transgenic animals.

[0100] After standard identification and breeding to create animalscarrying the transgene in all tissues, the baseline expression of thetet-operator linked transgene can be examined in various tissues ofinterest (e.g., by conventional techniques for analyzing mRNAexpression, such as Northern blotting, S1 nuclease mapping or reversetranscriptase-polymerase chain reaction). Additionally, basal expressionof the transgene can be examined in primary cultures of cells derivedfrom various tissues of the animal (e.g, skin cells in culture).

[0101] A second criterion for the selection of the stable clone is theability of the tet operator-linked transgene to respond to transient orstable expression of tTA upon transfection of a tTA expression plasmidlike pUHD15-1 or pUHD151-1. As will be appreciated, these plasmids arecited as examples only and others can be devised that expressedsufficient quantities of tTA in ES cells. The ability of tTA to induceexpression of a tet-operator linked transgene stably transfected into anES cell clone can be examined by supertransfecting the ES cell clonewith a tTA expression plasmid and assaying expression of the transgene.Alternatively, inducibility of a tet-operator linked transgene can beexamined in cells derived from various tissues of a transgenic animalcarrying the transgene by preparing primary cultures of cells from theanimal (e.g., skin cell cultures), transfecting the cells with a tTAexpression plasmid and assaying expression of the transgene in the cellsby standard techniques.

[0102] A clone fulfilling the criteria discusssed above is selected andexpanded in number. This clone is then used as a recipient of a geneknock-out procedure consisting of the following steps:

[0103] (1) flanking the sequences of a polynucleotide molecule encodinga tTA of the invention by DNA sequences from a second gene of interestsuch that the DNA sequences that normally control the expression of thesecond target gene of interest are fused to and control expression ofthe DNA sequences of encoding the tTA;

[0104] (2) introducing this chimeric gene into an embryonic cell linefrom a species of interest and modified as described above and screeningcandidate embryonic cell clones for those in which homologousrecombination has taken place at the locus of interest;

[0105] (3) introducing those recombinant cells into blastocysts from thespecies of interest; and

[0106] (4) implanting the chimeric embryo into the uteri ofpseudopregnant recipient mothers to facilitate development and birth ofa homologous recombinant animal.

[0107] This process results in offspring containing a replacement of theamino acid coding sequences of the second gene of interest with those ofthe DNA sequences of the tTA such that the tTA encoding sequence isexpressed in a temporal and spatial pattern similar to that of theendogenous second gene of interest. In this case, it is necessary toself cross the recombinant animals (or breed to homozygosity) so thatboth copies of the target sequence into which the tTA coding sequenceshave been integrated are interrupted. This procedure also leads tohomozygosity of the tet-operator linked transgene (i.e., animalshomozygous for both components of the genetic swith described herein canbe produced). These techniques are well established and described in:Wood et al. Proc. Natl. Acad. Sci. 90:45824585, Simon et al. NatureGenetics 1:92-97; and Soriano et al. Cell 64:693-702 and referencestherein.

[0108] In yet another embodiment of the invention, embryonic stem (ES)cell technology can again be used to prevent or promote expression of agene interest in a conditional manner using a single homologousrecombination step that will result in the integrated copy shown in FIG.13. In this method, a DNA construct containing a fusion of the sequencesthat normally flank the endogenous gene of interest at the 5′ end (andcontain sequences commonly referred to as promoter sequences) are fusedto the DNA sequences encoding the tTA molecule. At the 3′ end of the tTAcoding sequence, DNA sequences encoding resistance to a selectablemarker are typically included. For example, a neomycin resistance gene,which may be fused to either a constitutive regulatory element (e.g., apPGK promoter as depicted in FIG. 13A) or to a tet operator sequence(s)(as depicted in FIG. 13B) can be inserted at the 3′ end of the tTAencoding sequence. When the selectable marker is operably linked to atet operator sequence(s), its expression is regulated by the tTA (e.g.,a resistance phenotype will be expressed in the absence but not thepresence of Tc). Finally, 3′ of the selectable marker sequences in thisDNA construct are inserted the DNA sequences encoding the endogenousgene of interest, which are also fused to at least one tet operatorsequence and a minimal promoter.

[0109] Because in this configuration of the DNA molecule, conventionallycalled the targeting vector, the coding sequences of the tTA, theselectable marker and the endogenous gene of interest are all flanked bythe sequences normally flanking the endogenous gene of interest, thisDNA construct has the potential for homologous recombination with thelocus of the endogenous gene of interest upon its introduction intocells such as, but not limited to ES cells. Homologous recombination ofthis type alters the natural locus such that the gene of interest fallsunder the control of the tTA and consequently under regulation by thepresence or absence of tetracycline or derivative thereof. Theexpression of the tTA protein, on the other hand, follows the normalpattern of expression of the gene of interest.

[0110] Recombinant ES cells of this type are then used to generateintact organisms as has been described (Wood et al. Proc. Natl. Acad.Sci. 90:4582-4585, Simon et al. Nature Genetics 1:92-97; and Soriano etal. Cell 64:693-702) which can in turn be breed to homozygosity.

[0111] As will be appreciated, the close proximity of the promoterelements in this particular configuration of the DNA construct used forhomologous recombination may require special consideration to insulatethe downstream tet operator/minimal promoter operably linked to theendogenous gene from long range effects of the endogenous promoteroperably linked to the tTA coding sequence to achieve the required lowbasal level expression of the endogenous gene. Some possible solutionsare strong transcriptional terminators known to those of ordinary skillin the art, DNA elements that increase the distance between the elementsor others that limit the effect of enhancer sequences (e.g.,transcriptional insulators, including matrix attachment regions), all ofwhich are to be cloned alone or in combination in between the selectablemarker expression unit (e.g., neomycin resistance gene with linkedpromoter) and the tTA-responsive transcriptional promoter sequence (seeFIG. 13). Examples of suitable transcriptional terminators,transcriptional insulators, matrix attachment regions and/or othersequences which can be included in the “single hit” targeting vector toinhibit basal transcription of the tet operator-linked endogenous geneare described in Sato, K. et al. (1986) Mol. Cell. Biol. 6:1032-1043;Michel, D. et al. (1993) Cell. Mol. Biol. Res. 39:131-140; Chung, J. H.et al. (1993) Cell 74:505-514; Neznanov, N. et al. (1993) Mol. Cell.Biol. 13:2214-2223; and Thorey, I. S. et al. (1993) Mol. Cell. Biol.13:6742-6751.

[0112] The different animals resulting from any of the above mentionedembodiments can be studied either in the absence (endogenous geneswitched “on”) or presence (endogenous gene switched “off”) oftetracycline or tetracycline analogues as described for other transgenicanimals below. Such animals can be used to identify, compare andcharacterize the activity of substances which interact with, upon orthrough the action of the gene product of interest.

[0113] The present invention relates to a control system that ineucaryotic cells allows regulation of expression of an individual geneover up to five orders of magnitude. This system is based on regulatoryelements of a tetracycline resistance operon, e.g. Tn10 of E. coli(Hillen & Wissmann, “Topics in Molecular and Structural Biology,” inProtein-Nucleic Acid Interaction, Saeger & Heinemann, eds., Macmillan,London, 1989, Vol. 10, pp. 143-162), in which transcription ofresistance-mediating genes is negatively regulated by the tetracyclinerepressor (tetR). In the presence of tetracycline or a tetracyclineanalogue, tetR does not bind to its operators located within thepromoter region of the operon and allows transcription. By combiningtetR with a protein capable of activating transcription in eucaryotes,e.g. the C-terminal domain of VP16 from HSV (known to be essential forthe transcription of the immediate early vital genes (Triezenberg etal., (1988) Genes Dev. 2:718-729), a hybrid transactivator is generatedthat stimulates minimal promoters fused to tetracycline operator (tetO)sequences. These promoters are virtually silent in the presence of lowconcentrations of tetracycline, which prevents thetetracycline-controlled transactivator (tTA) from binding to tetOsequences.

[0114] The specificity of the tetR for its operator sequence (Hillen &Wissmann, “Topics in Molecular and Structural Biology,” inProtein-Nucleic Acid Interaction, Saeger & Heinemann, eds., Macmillan,London, 1989, Vol. 10, pp. 143-162) as well as the high affinity oftetracycline for tetR (Takahashi et al., J. Mol. Biol. 187:341-348(1986)) and the well-studied chemical and physiological properties oftetracyclines constitute a basis for an inducible expression system ineucaryotic cells far superior to the lacR/O/IPTG system. This hasalready been demonstrated in plant cells, in which direct repressoraction at promoter sites is efficiently reversed by the antibiotic (Gatz& Quail, (1988) Proc. Natl. Acad. Sci. USA 85:1394-1397, Gatz et al.,(1991) Mol. Gen. Genet. 227:229-237). However, these previous systemsused a tet repressor alone to inhibit gene expression, which may beinefficient or require high concentrations of the repressorintracellularly to function effectively. In contrast, the tTA of thepresent invention functions as a transcriptional activator to stimulateexpression of a tet operator-linked gene.

[0115] In particular, the invention relates to a polynucleotide moleculecoding for a transactivator fusion protein comprising the tet repressor(tetR) and a protein capable of directly or indirectly activatingtranscription in eucaryotes. The portion of the polynucleotide moleculecoding for tetR may be obtained according to Altschmied et al., EMBO J.7:4011-4017 (1988), the contents of which are fully incorporated byreference herein. Other tetR sequences are available from Genbank and/orare disclosed in Waters, S. H. et al. (1983) Nucl. Acids Res.11:6089-6105; Unger, B. et al. (1984) Gene 31:103-108, Unger, B. et al.(1984) Nucl Acids Res. 12:7693-7703; Tovar, K. et al. (1988) Mol. Gen.Genet. 215:76-80; Hillen, W. and Schollmeier, K. (1983) Nucl. Acids Res.11:525-539 and Postle, K. et al. (1984) Nucl. Acids Res. 12:4849-4863,the contents of each of which are fully incorporated herein byreference.

[0116] The portion of the polynucleotide molecule coding for thenegatively charged C-terminal domain of HSV-16, a protein known to beessential for transactivation in eucaryotes, may be obtained accordingto Triezenberg et al., Genes Dev. 2:718-729 (1988), the contents ofwhich are fully incorporated by reference herein. Preferably, theactivating domain comprises the C-terminal 130 amino acids of the virionprotein 16. Alternativly, other polypeptides with transcriptionalactivation ability in eucaryotic cells can be used in the tTA of theinvention. Transcriptional activation domains found within variousproteins have been grouped into categories based upon similar structuralfeatures. Types of transcriptional activation domains include acidictranscription activation domains, proline-rich transcription activationdomains, serine/threonine-rich transcription activation domains andglutamine-rich transcription activation domains. Examples of acidictranscriptional activation domains include the VP16 regions alreadydescribed and amino acid residues 753-881 of GAL4. Examples ofproline-rich activation domains include amino acid residues 399-499 ofCTF/NF1 and amino acid residues 31-76 of AP2. Examples ofserine/threonine-rich transcription activation domains include aminoacid residues 1-427 of ITF1 and amino acid residues 2-451 of ITF2.Examples of glutamine-rich activation domains include amino acidresidues 175-269 of Oct1 and amino acid residues 132-243 of Sp1. Theamino acid sequences of each of the above described regions, and ofother useful transcriptional activation domains, are disclosed inSeipel, K. et al. (EMBO J. (1992) 13:4961-4968).

[0117] The polynucleotide molecule coding for tetR may be linked to apolynucleotide molecule coding for the activating domain (e.g., of HSVVP16) and recombined with vector DNA in accordance with conventionalrecombinant DNA techniques, including blunt-ended or stagger-endedtermini for ligation, restriction enzyme digestion to provideappropriate termini, filling in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andligation with appropriate ligases. Alternatively, nucleic acid fragmentsencoding the repressor and the activating domain can be obtained bypolymerase chain reaction amplification of appropriate nucleotidesequences using template DNA encoding either the repressor or theactivating domain (e.g., encoding VP16). The amplified DNA fragments canthen be ligated such that the protein coding sequences remain in-frameand the chimeric gene so produced can be cloned into a suitableexpression vector.

[0118] Preferably, the polynucleotide molecule coding for thetransactivator fusion protein further comprises an operably linkedpromotor. The promotor may be an inducible promotor or a constitutivepromotor. Examples of such promotors include the human cytomegaloviruspromotor IE as taught by Boshart et al., Cell 41:521-530 (1985),ubiquitously expressing promotors such as HSV-Tk (McKnight et al., Cell37:253-262 (1984)) and β-actin promoters (e.g. the human P-actinpromoter as described by Ng et al., Mol. Cell. Biol. 5:2720-2732(1985)), as well as promoters in combination with control regionsallowing integration site independent expression of the transgene(Grosveld et al., Cell 51:975-985 (1987)), as well as tissue specificpromoters such as albumin (liver specific, Pinkert et al., Genes Dev.1:268-277 (1987)), lymphoid specific promoters (Calame and Eaton, Adv.Immunol. 43:235-275 (1988)), in particular promoters of T-cell receptors(Winoto and Baltimore, EMBO J. 8:729-733 (1989)) and immunoglobulins;Banerji et al., Cell 33:729-740 (1983); Queen and Baltimore, ibid.741-748), neuron specific promoters (e.g. the neurofilament promoter;Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86:5473-5477 (1989)),pancreas specific promoters (Edlund et al., Science 230:912-916 (1985))or mammary gland specific promoters (milk whey promoter, U.S. Pat. No.4,873,316 and European Application Publication No. 264,166) as well asdevelopmentally regulated promoters such as the muring hox promoters(Kessel and Cruss, Science 249:374-379 (1990)) or the α-fetoproteinpromoter (Campes and Tilghman, Genes Dev. 3:537-546 (1989)), thecontents of each of which are fully incorporated by reference herein.Preferably, the promoter is constitutive in the respective cell types.In one embodiment of the invention, the polynucleotide molecule encodingthe transactivator is integrated at a predetermined location within asecond target DNA molecule (e.g., a gene of interest within achromosome) such that the tTA-coding sequences are placed under thecontrol of endogeneous regulatory elements (e.g., a 5′ regulatory regionof a target gene of interest into which the tTA-coding sequence isintegrated). Depending upon which gene the tTA-coding sequences areintegrated into, the endogenous regulatory elements may provideconstitutive expression of the tTA in many cell types or may limitexpression of the tTA to a particular cell or tissue type.

[0119] The invention also relates to another polynucleotide moleculecoding for a protein, wherein said polynucleotide is operably linked toa tTA-responsive promoter. Typically, this tTA-responsive promotercomprises a minimal promotor operatively linked to at least one tetoperator (tetO) sequence. The tetO sequence may be obtained, forexample, according to Hillen & Wissmann, “Topics in Molecular andStructural Biology,” in Protein-Nucleic Acid Interaction, Saeger &Heinemann, eds., Macmillan, London, 1989, Vol. 10, pp. 143-162, thecontents of which are fully incorporated by reference herein. Other tetOsequences which may be used in the practice of the invention may beobtained from Genbank and/or are disclosed in Waters, S. H. et al.(1983) Nucl. Acids Res. 11:6089-6105; Hillen, W. and Schollmeier, K.(1983) Nucl. Acids Res. 11:525-539; Stuber, D. and Bujard, H. (1981)Proc. Natl. Acad. Sci. USA 78:167-171; Unger, B. et al. (1984) NuclAcids Res. 12:7693-7703; and Tovar, K. et al. (1988) Mol. Gen. Genet.215:76-80, which are fully incorporated by reference herein in theirentirety. One, two, three, four, five, six, seven, eight, nine or ten ormore copies of the tet operator sequence may be employed, with a greaternumber of such sequences allowing an enhanced range of regulation. Asshown in the Examples, multiple copies of the tet operator sequenceprovides a synergistic effect on the ability to control expression ofthe heterologous protein.

[0120] The polynucleotide sequence specifying the cytomegaloviruspromotor may be obtained according to Boshart et al., Cell 41:521-530(1985), the contents of which are fully incorporated by referenceherein. Preferably, positions +75 to −53 to +75 to −31 of thepromotor-enhancer are employed as a minimal promoter. The promotor maybe followed by a polylinker and then by the gene coding for the proteinof interest. While the luciferase gene or other reporter gene, e.g. thegene coding for chloramphenicol acetyltransferase or β-galactosidase,may be used to demonstrate the operability of the regulatory system, theinvention is not intended to be so limited. Examples of such genesinclude, but are not limited to the estrogen receptor, the GABAreceptor, the progesterone receptor and the X-protein of HBV.

[0121] The present invention also relates to eucaryotic cellstransfected with the polynucleotide molecules of the present invention.In particular, the invention relates to eucaryotic cells transfectedwith

[0122] (a) a first polynucleotide molecule coding for a transactivatorfusion protein comprising a prokaryotic tet repressor and a proteincapable of activation transcription in eucaryotes; and

[0123] (b) a second polynucleotide molecule coding for a protein,wherein said second polynucleotide molecule is operably linked to aminimal promotor and at least one tet operator sequence.

[0124] The two polynucleotide molecules may reside on the same orseparate vectors. In a preferred embodiment, the first polynucleotide isintegrated into the chromosome of a eucaryotic cell or transgenic animaland the second polynucleotide is introduced as part of a vector.Integration may be achieved where there is crossover at regions ofhomology shared between the incoming polynucleotide molecule and theparticular genome.

[0125] The expression of the heterologous protein from such transfectedeucaryotic cells may be tightly regulated. Unexpectedly, it has beendetermined that the expression system of the present invention may beused to regulate expression by about 5 orders of magnitude. In addition,it has been discovered that the expression system of the presentinvention allows one to rapidly turn “on” and “off” the expression ofthe heterologous gene in a reversible way. Moreover, it has beendiscovered that the expression system of the invention allows one toachieve a desired level of expression according to how much tetracyclineor tetracycline analogue is employed (see FIG. 3). Thus, the expressionsystem of the present invention is is a great advance in the art.

[0126] The invention also relates to transgenic animals comprising atleast a first polynucleotide molecule of the present invention encodinga tTA. Such transgenic animals may be obtained, for example, byinjecting the polynucleotide into a fertilized egg which is allowed todevelop into an adult animal. In particular, a few hundred DNA moleculesare injected into the pro-nucleus of a fertilized one cell egg. Themicroinjected eggs are then transferred into the oviducts ofpseudopregnant foster mothers and allowed to develop. It has beenreported by Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442(1985), the contents of which are fully incorporated by referenceherein, that about 25% of mice which develop will inherit one or morecopies of the microinjected DNA. It is also possible to prepare apolynucleotide molecule comprising a milk protein promotor andmicroinject the DNA into the fertilized egg to give, upon development, atransgenic mammal which is capable of producing the heterologous proteinin its milk, when in the absence of tetracycline or a tetracyclineanalog. See International Application Publication No. WO 88/00239 andEuropean Application Publication No. 0264,166, the contents of which arefully incorporated by reference herein.

[0127] The invention also relates to non-human animals and theiroffspring derived by homologous recombination of the DNA sequences ofthe first polynucleotide molecules of the invention into a specific DNAsite containing the nucleotide sequences of a gene referred to as thetarget gene. This would be accomplished in the following steps: 1)flanking the sequences of the first polynucleotide molecule of theinvention encoding a tTA by DNA sequences from the target site such thatthe DNA sequences that normally control the expression of the targetgene are fused to and control the expression of the DNA sequences of thefirst polynucleotide molecules of the invention, 2) introducing thischimeric gene into an embryonic cell line from the species of interestand screening candidate embryonic cell clones for those in whichhomologous recombination has taken place at the target gene locus, 3)introducing those recombinant cells into a blastocysts from the speciesof interest, 4) implantating the chimeric embryo into the uteri ofpseudopregnant recipient mothers to facilitate development and birth.This process will result in offspring containing a replacement of theamino acid coding sequences of the target gene with those of the DNAsequences of the first polynucleotide molecule of the invention suchthat this corresponding amino acid sequence will be expressed in apattern similar to that of the target gene. These processes and theirresults are collectively and commonly referred to as “gene knock-out”.

[0128] These techniques are well established and described in: Wood etal. Proc. Natl. Acad. Sci 90:4582-4585, Simon et al. Nature Genetics1:92-97 & Soriano et al. Cell 64:693-702 and references therein.

[0129] The invention also relates to a method to prevent or promote theexpression of the target gene in a conditional manner. This may beaccomplished by breeding an animal containing the target gene knock-out(as outlined in the preceding paragraph) with a transgenic animalderived by the following method. The transgenic animal would beconstructed by inserting, by micro-injection, a chimeric DNA sequence(commonly referred to as a chimeric transgene) consisting of the DNAsequences of the second polynucleotide molecule of the inventioninserted 5′ of the DNA sequences encoding the amino acid sequence of thetarget gene into the genome of a fertilized egg which is allowed todevelop into an adult animal. The protocol for the construction of suchtransgenic animals is a well established technique (Brinster et al.Proc. Natl. Acad. Sci. 83:4432-4445, Crenshaw et al. Genes & Dev3:959-972 and references therein) as is the breeding of animals. Fromthe breeding will result offspring containing both the gene knock-outand the chimeric transgene. That is, replacement of the amino acidcoding sequences of the target gene with those of the DNA sequences ofthe first polynucleotide molecule of the invention such that thiscorresponding amino acid sequence will be expressed in a pattern similarto that of the target gene and, the DNA sequences of the secondpolynucleotide molecule of the invention inserted 5′ of the DNAsequences encoding the amino acid sequence of the target gene. In thiscombination the target gene can be regulated by the addition orsubstraction of tetracycline or its analogs from the food or watersupply of the animal.

[0130] Thus, the invention also relates to a method to down regulate theexpression of a protein coded for by a polynucleotide, comprisingcultivating the transfected eucaryotic cells of the present invention ina medium comprising tetracycline or a tetracycline analogue. Asdescribed in the Examples, it is possible to closely control the extentof expression by carefully controlling the concentration of tetracyclineor tetracycline analogue in the culture media. As shown in FIG. 3, panelA, as little as 0.0001 μg/ml of tetracycline will begin to result in adecrease of polypeptide (luciferase) expression. At about 0.1 μg/ml, theexpression is essentially shut off. The concentration of tetracycline ortetracycline analogue which can be used to regulate the expression levelmay range from about 0.001 to about 1 μg/ml.

[0131] The invention also relates to a method to up regulate theexpression of a protein coded for by a polynucleotide, comprisingcultivating the eucaryotic cells of the present invention in a mediumlacking tetracycline or a tetracycline analogue.

[0132] The invention also relates to a method to use regulated geneexpression in the production of recombinant proteins as generallyreviewed by Yarranton, G. T 1992, the whole article incorporated asreference herein. Expression of recombinant proteins that are cytotoxicor otherwise infer with physiological processes in cells has beenhampered by the lack of suitable methods to tightly regulate geneexpression. In contrast, a production cell line according to the currentinvention is grown in the presence of tetracycline or tetracyclineanalogues until an optimal density (assessed empirically to allow forsubsequent induction of gene expression) and expression is induced bydilution of the regulating compound. The culture is continuosly grownuntil an optimal expression level has been reached. The recombinantprotein is then harvested according to standard procedures.

[0133] As a preferred embodiment, eucaryotic cells are used forexpression of recombinant proteins as generally reviewed in “GeneTransfer and Expression” (M. Kriegler 1990) incorporated herein asreference. While CHO^(dhfr-) cells (Urlaub, G. and Chasin, L. 1980),293cells (Graham, F.L. et al. 1977) or myeloma cells like SP2 or NSO(Galfre, C. and Milstein, C. 1981) are commonly used it should be clearto the skilled in the art, that any eucaryotic cell line can be usedthat is suitable for the protein to be expressed, the selection systemchosen and the fermentation system employed.

[0134] In another preferred embodiment, the cells used for regulatedexpression are yeast cells including, but not limited to Saccharomycescerevisiae, Pichia pastoris, Kluyveromyces lactis and Hansenulapolymorpha as generally reviewed by Fleer, R. 1992, the whole articleincorporated as referenced herein. In another preferred embodiment, thecells used for regulated expression are insect cells with the gene andpromoter region carried on the baculovirus genome as generally reviewedin “Baculovirus expression vectors” (O′Reilly et al. 1992), the wholedocument incorporated as referenced herein.

[0135] As can be appreciated, the tissue specificity of some promotersdictate that the tet operator sequence/promoter sequence fusion has tobe designed with the particular application and cell line in mindfollowing the teachings in this application using the promoterscustomarily used for the cell line in question; examples for thosepromoters are given in the relevant references mentioned above.

[0136] It should be clear from the foregoing that it is critical in thecurrent invention that the production cell line is selected for a verylow basal expression of the gene under control of the Tet operator/CMVpromoter sequence. There are numerous methods currently availableemploying enzymatically assisted or unassisted homologous recombinationto target repeatedly a chromosal location found empirically to be suitedfor the integration of the gene encoding the recombinant protein. Inaddition to the homologous recombination approaches already describedherein, enzyme-assisted site-specific integration systems are known inthe art and can be applied to the components of the regulatory system ofthe invention to integrate a DNA molecule at a predetermined location ina second target DNA molecule. Examples of such enzyme-assistedintegration systems include the Cre recombinase-lox target system (e.g.,as described in Baubonis, W. and Sauer, B. (1993) Nucl. Acids Res.21:2025-2029; and Fukushige, S. and Sauer, B. (1992) Proc. Natl. Acad.Sci. USA 89:7905-7909) and the FLP recombinase-FRT target system (e.g.,as described in Dang, D. T. and Perrimon, N. (1992) Dev. Genet.13:367-375; and Fiering, S. et al. (1993) Proc. Natl. Acad. Sci. USA90:8469-8473).

[0137] Media which may be used in the practice of the invention includeany media which are compatible with the transfected eucaryotic cells ofthe present invention. Such media are commercially available (e.g. fromGibco/BRL).

[0138] Alternatively, it is possible to down regulate the expression ofa protein in a transgenic animal of the present invention byadministering to the animal tetracycline or tetracycline analogue. Thetetracycline or tetracycline may be administered by any means thatachieves its intended purpose, e.g. by parenteral, subcutaneous,intravenous, intramuscular, intraperitoneal, transdermal, or buccalroutes. Alternatively, or concurrently, administration may be by theoral route (see e.g., Example 2). The dosage administered will bedependent upon the age, health, and weight of the animal, kind ofconcurrent treatment, if any, and frequency of treatment. To up regulatethe expression of the protein, the administration of tetracycline ortetracycline analogue may then be interrupted.

[0139] The invention also relates to a kit comprising a carrier meanshaving in close confinement therein at least two container means such astubes, vials, bottles and the like, each of which containing apolynucleotide molecule which can be used in the practice of theinvention. In particular, the invention relates to a kit comprising acarrier means having in close confinement therein at least two containermeans, wherein a first container means contains a first polynucleotidemolecule coding for a transactivator fusion protein comprising aprokaryotic tet repressor and a protein capable of activationtranscription in eucaryotes in a form suitable for homologousrecombination; and a second container means contains a secondpolynucleotide molecule comprising a minimal promotor operably linked toat least one tet operator sequence, wherein the second polynucleotidemolecule is capable of being ligated to a heterologous gene sequencecoding for a polypeptide and activating the expression of theheterologous protein.

[0140] The invention also relates to kits comprising a carrier meanshaving in close confinement therein at least two container means,wherein a first container means contains a eucaryotic cell transfectedwith a first polynucleotide molecule coding for a transactivator fusionprotein comprising a prokaryotic tet repressor and a protein capable ofactivation transcription in eucaryotes in a form suitable for homologousrecombination; and a second container means contains a secondpolynucleotide molecule comprising a minimal promotor operably linked toat least one tet operator sequence, wherein the second polynucleotidemolecule is capable of being ligated to a heterologous gene sequencecoding for a polypeptide and activating expression of the polypeptide.

[0141] The invention is widely applicable to a variety of situationswhere it is desirable to be able to turn gene expression “on” and “off”,or regulate the level of gene expression, in a rapid, efficient andcontrolled manner without causing pleiotropic effects or cytotoxicity.The invention may be particularly useful for gene therapy purposes inhumans, in treatments for either genetic or acquired diseases. Thegeneral approach of gene therapy involves the introduction of one ormore nucleic acid molecules into cells such that one or more geneproducts encoded by the introduced genetic material are produced in thecells to restore or enhance a functional activity. For reviews on genetherapy approaches see Anderson, W. F. (1992) Science 256:808-813;Miller, A. D. (1992) Nature 357:455-460; Friedmann, T. (1989) Science244:1275-1281; and Cournoyer, D., et al. (1990) Curr. Opin. Biotech.1:196-208. However, current gene therapy vectors typically utilizeconstitutive regulatory elements which are responsive to endogenoustranscriptions factors. These vector systems do not allow for theability to modulate the level of gene expression in a subject. Incontrast, the regulatory system of the invention provides this ability.

[0142] To use the system of the invention for gene therapy purposes, atleast one DNA molecule is introduced into cells of a subject in need ofgene therapy (e.g., a human subject suffering from a genetic or acquireddisease) to modify the cells. The cells are modified to contains 1)nucleic acid encoding a tTA of the invention in a form suitable forexpression of the tTA in the host cells and 2) a gene of interest (e.g.,for therapeutic purposes) operatively linked to a tTA-responsivepromoter (e.g., a tet operator sequence(s) and minimal promoter).Preferably, one or both of these DNA molecules is integrated into apredetermined location within a chromosome of the human cells byhomologous recombination. A single DNA molecule encoding both componentsof the regulatory system of the invention can be used, or alternatively,separate DNA molecules encoding each component can be used. The cells ofthe subject can be modified ex vivo and then introduced into the subjector the cells can be directly modified in vivo by conventional techniquesfor introducing nucleic acid into cells. Expression of the gene ofinterest in the cells of the subject is stimulated in the absence of Tcor a Tc analogue, whereas expresion is then inhibited by administeringTc or a Tc analogue to the patient. The level of gene expression can bevaried depending upon which particular Tc analogue is used as theinducing agent. Additionally, expression of the gene of interest can beadjusted according to the medical needs of the individual, which mayvary throughout the lifetime of the individual. Thus, the regulatorysystem of the invention offers the advantage over constitutiveregulatory systems of allowing for modulation of the level of geneexpression depending upon the requirements of the therapeutic situation.

[0143] Genes of particular interest to be expressed in cells of asubject for treatment of genetic or acquired diseases include thoseencoding adenosine deaminase, Factor VIII, Factor IX, dystrophin,β-globin, LDL receptor, CFTR, insulin, erythropoietin, anti-angiogenesisfactors, growth hormone, glucocerebrosidase, β-glucouronidase,α1-antitrypsin, phenylalanine hydroxylase, tyrosine hydroxylase,ornithine transcarbamylase, arginosuccinate synthetase, UDP-glucuronysyltransferase, apoA1, MDR1 and MRP multidrug resistance genes, TNF,soluble TNF receptor, interleukins (e.g., IL-2), interferons (e.g., α-or γ-IFN) and other cytokines and growth factors.

[0144] Gene therapy applications of particular interest in cancertreatment include overexpression of a cytokine gene (e.g., TNF-α) intumor infiltrating lymphocytes or ectopic expression of cytokines intumor cells to induce an anti-tumor immune response at the tumor site),expression of an enzyme in tumor cells which can convert a non-toxicagent into a toxic agent, expression of tumor specific antigens toinduce an anti-tumor immune response, expression of tumor suppressorgenes (e.g., p53 or Rb) in tumor cells, expression of a multidrugresistance gene (e.g., MDR1 and/or MRP) in bone marrow cells to protectthem from the toxicity of chemotherapy.

[0145] Gene therapy applications of particular interest in treatment ofviral diseases include expression of trans-dominant negative viraltransactivation proteins, such as trans-dominant negative tat and revmutants for HIV or trans-dominant ICp4 mutants for HSV (see e.g.,Balboni, P. G. et al. (1993) J. Med. Virol. 41:289-295; Liem, S. E. etal. (1993) Hum. Gene Ther. 4:625-634; Malim, M. H. et al. (1992) J. Exp.Med. 176:1197-1201; Daly, T. J. et al. (1993) Biochemistry 32:8945-8954;and Smith, C. A. et al. (1992) Virology 191:581-588), expression oftrans-dominant negative envelope proteins, such as env mutants for HIV(see e.g., Steffy, K. R. et al. (1993) J. Virol. 67:1854-1859),intracellular expression of antibodies, or fragments thereof, directedto viral products (“internal immunization”, see e.g., Marasco, W. A. etal. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893) and expression ofsoluble viral receptors, such as soluble CD4.

[0146] The regulatory system of the invention can also be used toexpress a suicide gene (such as a ricin or HSV tk gene) in cells in aconditional manner to allow for destruction of the cells (e.g., in vivo)following a particular therapy. For example, a suicide gene can beintroduced into tumor cells to be used for anti-cancer immunization orinto the viral genome of a live attenuated viral to be used as avaccine. The tumor cells or viral vaccine carrying the suicide gene areadministered to a subject in the presence of Tc (or analogue thereof).

[0147] Following immunization, the drug is withdrawn (e.g.,administration is stopped), thereby inducing expression of the suicidegene to destroy the tumor cells or cells carrying the live virus.

[0148] Cells types which can be modified for gene therapy purposesinclude hematopoietic stem cells, myoblasts, hepatocytes, lymphocytes,airway epithelium and skin epithelium. For further descriptions of celltypes, genes and methods for gene therapy see e.g., Wilson, J. M. et al.(1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano, D. et al.(1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Wolff, J. A. et al.(1990) Science 247:1465-1468; Chowdhury, J. R. et al. (1991) Science254:1802-1805; Ferry, N. et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Wilson, J. M. et al. (1992) J Biol. Chem. 26:963-967;Quantin, B. et al. ((1992) Proc. Natl. Acad. Sci. USA 89:2581-2584; Dai,Y. et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; vanBeusechem, V. W. et al. (1992) Proc. Natl. Acad Sci. USA 89:7640-7644;Rosenfeld, M. A. et al. (1992) Cell 68:143-155; Kay, M. A. et al. (1992)Human Gene Therapy 3:641-647; Cristiano, R. J. et al. (1993) Proc. Natl.Acad. Sci. USA 90:2122-2126; Hwu, P. et al. (1993) J Immunol.150:4104-4115; and Herz, J. and Gerard, R. D. (1993) Proc. Natl. Acad.Sci. USA 90:2812-2816.

[0149] The Tc-controlled regulatory system of the invention has numerousadvantages properties that it particularly suitable for application togene therapy. For example, the system provides an “on”/“off ” switch forgene expression that allows for regulated dosaging a gene product in asubject. There are several situations in which it may be desirable to beable to provide a gene product at specific levels and/or times in aregulated manner, rather than simply expressing the gene productconstitutively at a set level. For example, a gene of interest can beswitched “on” at fixed intervals (e.g., daily, alternate days, weekly,etc.) to provide the most effective level of a gene product of interestat the most effective time. The level of gene product produced in asubject can be monitored by standard methods (e.g., direct monitoringusing an immunological assay such as ELISA or RIA or indirectly bymonitoring of a laboratory parameter dependent upon the function of thegene product of interest, e.g., blood glucose levels and the like). Thisability to turn “on” expression of a gene at discrete time intervals ina subject while also allowing for the gene to be kept “off” at othertimes avoids the need for continued administration of a gene product ofinterest at intermittent intervals. This approach avoids the need forrepeated injections of a gene product, which may be painful and/or causeside effects and would likely require continuous visits to a physician.In contrast, the system of the invention avoids these drawbacks.Moreover, the ability to turn “on” expression of a gene at discrete timeintervals in a subject allows for focused treatment of diseases whichinvolve “flare ups” of activity (e.g., many autoimmune diseases) only attimes when treatment is necessary during the acute phase when pain andsymptoms are evident. At times when such diseases are in remission, theexpression system can be kept in the “off” state.

[0150] Gene therapy applications that may particularly benefit from thisability to modulate gene expression during discrete time intervalsinclude the following non-limiting examples:

[0151] Rheumatoid arthritis-genes which encode gene products thatinhibit the production of inflammatory cytokines (e.g., TNF, IL-1 andIL-12). can be expressed in subjects. Examples of such inhibitorsinclude soluble forms of a receptor for the cytokine. Additionally oralternatively, the cytokines IL-10 and/or IL-4 (which stimulate aprotective Th2-type response) can be expressed. Moreover, aglucocorticomimetic receptor (GCMR) can be expressed.

[0152] Hypopituitarism-the gene for human growth hormone can beexpressed in such subjects only in early childhood, when gene expressionis necessary, until normal stature is achieved, at which time geneexpression can be downregulated.

[0153] Wound healing/Tissue regeneration-Factors (e.g., growth factors,angiogenic factors, etc.) necessary for the healing process can beexpressed only when needed and then downregulated.

[0154] Anti-Cancer Treatments-Expression of gene products useful inanti-cancer treatment can be limited to a therapeutic phase untilretardation of tumor growth is achieved, at which time expression of thegene product can be downregulated. Possible systemic anti-cancertreatments include use of tumor infiltrating lymphocytes which expressimmunostimulatory molecules (e.g., IL-2, IL-12 and the like),angiogenesis inhibitors (PF4, IL-12, etc.), Herregulin, Leukoregulin(see PCT Publication No. WO 85/04662), and growth factors for bonemarrow support therapy, such as G-CSF, GM-CSF and M-CSF. Regarding thelatter, use of the regulatory system of the invention to express factorsfor bone marrow support therapy allows for simplified therapeuticswitching at regular intervals from chemotherapy to bone marrow supporttherapy (similarly, such an approach can also be applied to AIDStreatment, e.g., simplified switching from anti-viral treatments to bonemarrow support treatment). Furthermore, controlled local targeting ofanti-cancer treatments are also possible. For example, expression of asuicide gene by a regulator of the invention, wherein the regulatoritself is controlled by, for example, a tumor-specific promoter or aradiation-induced promoter.

[0155] In another embodiment, the regulatory system of the invention isused to express angiogenesis inhibitor(s) from within a tumor via atransgene regulated by the system of the invention. Expression ofangiogenesis inhibitors in this manner may be more efficient thansystemic administration of the inhibitor and would avoid any deleteriousside effects that might accompany systemic administration. Inparticular, restricting angiogenesis inhibitor expression to withintumors could be particularly useful in treating cancer in children stillundergoing angiogenesis associated with normal cell growth.

[0156] In another embodiment, high level regulated expression ofcytokines. may represent a method for focusing a patients own immuneresponse on tumor cells. Tumor cells can be transduced to expresschemoattractant and growth promoting cytokines important in increasingan individual's natural immune response. Because the highestconcentrations of cytokines will be in the proximity of the tumor, thelikelihood of eliciting an immunological response to tumor antigens isincreased. A potential problem with this type of therapy is that thosetumor cells producing the cytokines will also be targets of the immuneresponse and therefor the source of the cytokines will be eliminatedbefore eradication of all tumor cells can be certain. To combat this,expression of viral proteins known to mask infected cells from theimmune system can be placed under regulation, along with the cytokinegene(s), in the same cells. One such protein is the E19 protein fromadenovirus (see e.g., Cox, Science 247:715). This protein preventstransport of class I HLA antigens to the surface of the cell and henceprevents recognition and lysis of the cell by the host's cytotoxic Tcells. Accordingly, regulated expression of E19 in tumor cells couldshield cytokine producer cells from cytotoxic T cells during the onsetof an immune response provoked by cytokine expression. After asufficient period of time has elapsed to eradicate all tumor cells butthose expressing E19, E19 expression can be turned off, causing thesecells then to fall victim to the provoked anti-tumor immune response.

[0157] Benign prostatic hypertrophy-Similar to the above, a suicide genecan be regulated by a regulator of the invention, wherein the regulatoritself is controlled by, for example, a prostate-specific promoter.

[0158] The ability to express a suicide gene (e.g., an apoptosis gene,TK gene, etc) in a controlled manner using the regulatory system of theinvention adds to the general safety and usefulness of the system. Forexample, at the end of a desired therapy, expression of a suicide genecan be triggered to eliminate cells carrying the gene therapy vector,such as cells in a bioinert implant, cells that have disseminated beyondthe intended original location, etc. Moreover, if a transplant becomestumorous or has side effects, the cells can be rapidly eliminated byinduction of the suicide gene.

[0159] The regulatory system of the invention further offers the abilityto establish a therapeutically relevant expression level for a geneproduct of interest in a subject, in contrast to unregulatedconstitutive expression which offers no flexibility in the level of geneproduct expression that can be achieved. A physiologically relevantlevel of gene product expression can be established based on theparticular medical need of the subject, e.g., based on laboratory teststhat monitor relevant gene product levels (using methods as describedabove). In addition to the clinical examples and gene products alreadydiscussed above with gene to dosaging of the gene product, othertherapeutically relevant gene products which can be expressed at adesired level at a desired time include: Factor XIII and IX inhemophiliacs (e.g., expression can be elevated during times of risk ofinjury, such as during sports); insulin or amylin in diabetics (asneeded, depending on the state of disease in the subject, diet, etc.);erythropoietin to treat erythrocytopenia (as needed, e.g., at end-stagerenal failure); low-density lipoprotein receptor (LDLr) or verylow-density lipoprotein receptor (VLDLr) for artherosclerosis or genetherapy in liver (e.g, using ex vivo implants). Applications totreatment of central nervous system disorders are also encompassed. Forexample, in Alzheimer's disease, “fine tuned” expression of cholineacetyl transferase (CHAT) to restore acetylcholine levels, neurotrophicfactors (e.g., NGF, BDNGF and the like) and/or complement inhibitors(e.g., sCR1, sMCP, sDAF, sCD59 etc.) can be accomplished. Such geneproducts can be provided, for example, by transplanted cells expressingthe gene products in a regulated manner using the system of theinvention. Moreover, Parkinson's disease can be treated by “fine tuned”expression of tyrosine hydroxylase (TH) to increase levodopa anddopamine levels.

[0160] In addition to the proteinaceous gene products discussed above,gene products that are functional RNA molecules (such as anti-sense RNAsand ribozymes) can be expressed in a controlled manner in a subject fortherapeutic purposes. For example, a ribozyme can be designed whichdiscriminates between a mutated form of a gene and a wild-type gene.Accordingly, a “correct” gene (e.g., a wild-type p53 gene) can beintroduced into a cell in parallel with introduction of a regulatedribozyme specific for the mutated form of the gene (e.g., a mutatedendogenous p53 gene) to remove the defective mRNA expressed from theendogenous gene. This approach is particularly advantageous insituations in which a gene product from the defective gene wouldinterfere with the action of the exogenous wild-type gene.

[0161] Expression of a gene product in a subject using the regulatorysystem of the invention is modulated using tetracycline or analoguesthereof. Such drugs can be administered by any route appropriate fordelivery of the drug to its desired site of action (e.g., delivery tocells containing a gene whose expression is to be regulated). Dependingon the particular cell types involved, preferred routes ofadministration may include oral administration, intravenousadministration and topical administration (e.g., using a transdermalpatch to reach cells of a localized transplant under the skin, such askeratinocytes, while avoiding any possible side effects from systemictreatment).

[0162] In certain gene therapy situations, it may be necessary ordesirable to take steps to avoid or inhibit unwanted immune reactions ina subject receiving treatment. To avoid a reaction against the cellsexpressing the therapeutic gene product, a subject's own cells aregenerally used, when possible, to express the therapeutic gene product,either by in vivo modification of the subject's cells or by obtainingcells from the subject, modifying them ex vivo and returning them to thesubject. In situations where allogeneic or xenogeneic cells are used toexpress a gene product of interest, the regulatory system of theinvention, in addition to regulating a therapeutic gene, can also beused to regulate one or more genes involved in the immune recognition ofthe cells to inhibit an immune reaction against the foreign cells. Forexample, cell-surface molecules involved in recognition of a foreigncell by T lymphocytes can be downmodulated on the surface of a foreigncell used for delivery of a therapeutic gene product, such as byregulated expression in the foreign cell of a ribozyme which cleaves themRNA encoding the cell-surface molecule. Particularly preferred cellsurface molecules which can be downmodulated in this manner to inhibitan unwanted immune response include class I and/or class II majorhistocompatibility complex (MHC) molecules, costimulatory molecules(e.g., B7-1 and/or B7-2), CD40, and various “adhesion” molecules, suchas ICAM-1 or ICAM-2. Furthermore, as described above regardinganti-cancer treatments, a viral protein (e.g., adenovirus E19 protein)that downmodulates expression of MHC antigens can be regulated in hostcells using the system of the invention as a means of avoiding unwantedimmunological reactions.

[0163] In addition to avoiding or inhibiting an immune response againsta foreign cell delivering a therapeutic gene product, it may also benecessary, in certain situations, to avoid or inhibit an immune responseagainst certain components of the regulatory system of the invention(e.g., the regulator fusion proteins described herein) that areexpressed in a subject, since these fusion proteins containnon-mammalian polypeptides that may stimulate an unwanted immunereaction. In this regard, regulator fusion proteins can be designedand/or selected for a decreased ability to stimulate an immune responsein a host. For example, a transcriptional activator domain for use inthe regulator fusion protein can be chosen which has minimalimmunogenicity. In this regard, a wild-type transcriptional activationdomain of the herpes simplex virus protein VP 16 may not be a preferredtranscriptional activation domain for use in vivo, since it maystimulate an immune response in mammals. Alternative transcriptionalactivation domains can be used, as described herein, based on theirreduced immunogenicity in a subject. For example, a transcriptionalactivation domain of a protein of the same species as the host may bepreferred (e.g., a transcriptional activation domain from a humanprotein for use of a regulatory fusion protein in humans).Alternatively, a regulatory fusion protein of the invention can bemodified to reduce its immunogenicity in subjects, e.g., by identifyingand modifying one or more dominant T cell epitopes within a polypeptideof the fusion protein (e.g., either the Tet repressor moiety or thetranscriptional modulator moiety, such as a VP16 polypeptide). Such Tcell epitopes can be identified by standard methods and altered bymutagenesis, again by standard methods. A modified form of a regulatorfusion protein can then be selected which retains its originaltranscriptional regulatory ability yet which exhibits reducedimmunogenicity in a subject as compared to an unmodified fusion protein.

[0164] In addition to the foregoing, all conventional methods forgenerally or specifically downmodulating immune responses in subjectscan be combined with the use of the regulatory system of the inventionin situations where inhibition of immune responses is desired. Generalimmunosuppressive agents, such as cyclosporin A and/or FK506, can beadministered to the subject. Alternatively, immunomodulatory agentswhich may allow for more specific immunosuppression can be used. Suchagents may include inhibitors of costimulatory molecules (e.g., aCTLA4Ig fusion protein, soluble CD4, anti-CD4 antibodies, anti-B7-1and/or anti-B7-2 antibodies or anti-gp39 antibodies).

[0165] Finally, in certain situations, a delivery vehicle for cellsexpressing a therapeutic gene can be chosen which minimizes exposure oftransplanted cells to the immune system. For example, cells can beimplanted into bioinert capsules/biocompatible membranes with poreswhich allow for diffusion of proteins (e.g., a therapeutic gene productof interest) out of the implant and diffusion of nutrients and oxygeninto the implant but which prevent entry of immune cells, therebyavoiding exposure of the transplanted cells to the immune system (as hasbeen applied to islet cell transplantation).

[0166] Use of Conditional Knockout Animals as Models for Human Disease

[0167] The transgenic and conditional knockout animals of the inventionare also useful for creating animal models of human disease, inparticular for determining the role of a gene of interest in theprogression of a disease state. Conventional knockout technology, inwhich a gene is disrupted at an embryonic stage to produce an animal inwhich the gene product is never expressed, only allows one to evaluatethe role of the gene product in the initiation of a disease condition(e.g., the disease condition never develops in the animals). This systemsuffers from the limitation of yielding information valid only forevaluating the possible prophylactic effect of inhibiting the geneproduct. However, the conditional knockout system of the inventionallows one to evaluate the effect of inhibiting the expression of aparticular gene product on disease progression even after the diseasestate has been initiated. For example, a homologous recombinant animalcan be created in which an endogenous gene thought to be involved in theprogression of a disease state is operatively linked to at least one tetoperator sequence to confer tTA-mediated regulation on the gene. Thus,the gene is only expressed when tTA binds to the tet operator sequences,which occurs only in the absence of tetracycline. This animal can thenbe crossbred to a second animal transgenic for the tTA gene to createdouble transgenic animals in which, in the absence of tetracycline, tTAbinds to the tet operator-linked gene of interest to thereby keep thegene of interest turned “on” in the animals until it is desirable toturn the gene “off”. A disease state can then be induced in the doubletransgenic animals. After progression of the disease state for aninterval of time, expression of the tet operator linked gene of interestcan be turned “off” by administering tetracycline (or analogue) to theanimal. The effect of ablating expression of the gene product ofinterest after initiation of the disease state can thus be evaluated.This approach has the advantage that it yields valid informationregarding the value of inhibiting a gene product after the onset of adisease, a situation more closely resembling the typical therapeuticregimen in human disease.

[0168] In a non-limiting example of this approach of applying theconditional knockout system to the study of models of human diseasestates, a double transgenic animal as described above is created inwhich the gene for an interleukin-1-β converting enzyme (ICE), whichcleaves interleukin-1β (IL-1β) to its active form, is operatively linkedto at least one tet operator sequence. IL-1β is thought to be involvedin the progression of diseases such as septic shock, inflammatorydiseases and autoimmune diseases. Since ICE is necessary for theproduction of active IL-1β, one strategy for controlling IL-1β-mediateddisease states is to inhibit ICE activity. Accordingly, the effect ofinhibiting ICE on the progression of a disease state can be evaluated inthe double transgenic animals as follows. In the absence oftetracycline, expression of ICE in the double transgenic animals is kept“on” by the tTA.

[0169] Thus, in the absence of Tc, a disease condition can be inducedwhile ICE is still being expressed. For example, sepsis can be inducedin the animals by injection of lipopolysaccharide (LPS). After inductionof sepsis, tetracycline (or an analogue thereof) can be administered tothe animal to remove the tTA bound to the tet operator-linked ICE gene,thereby inhibiting the expression of the ICE gene. The effect ofinhibiting ICE expression after initiation of sepsis can thus beevaluated. Additional suitable applications of this approach to theregulation of other gene products thought to be involved in theprogression of a various disease states will be readily apparent to theskilled artisan.

[0170] Additional examples of genes which may be of particular interestfor regulation using the conditional knockout system of the inventioninclude cell cycle regulators. For example, the system can be used toevaluate the role of genes in regulating the progression of cellsthrough the cell cycle. Typically, aberrant or ectopic expression ofcell cycle regulators in cells is expected to lead to cell cycle arrestand, consequently, results in an inability to isolate cells expressingsuch cell cycle regulators. Accordingly, experimental expression of cellcycle regulators has been hindered by the lack of suitable methods fortightly regulating the expression of cell cycle regulators. In contrast,an experimental cell line in which a gene encoding a cell cycleregulator is controlled using the regulatory system of the invention canbe grown in the presence of tetracycline (or analogue) until inductionof gene expression is desired. More specifically, this regulatedexpression of cell cycle regulators may be useful in identifying genesand gene products important in enabling natural anti-cancer processes tooccur.

[0171] The regulatory system of the invention can also be used toproduce and isolate a gene product (e.g., protein) of interest. Largescale production of a protein of interest can be accomplished usingcultured cells in vitro which have been modified to contain 1) nucleicacid encoding a tTA of the invention in a form suitable for expressionof the tTA in the host cells and 2) a gene of interest (e.g., encoding aprotein of interest) operatively linked to a tTA-responsive promoter(e.g., a tet operator sequence(s) and minimal promoter). For example,mammalian, yeast or fungal cells can be modified to contain thesenucleic acid components as described herein. Alternatively, an insectcell/baculovirus expression system can be used. To produce and isolate agene product of interest, a host cell (e.g., mammalian, yeast or fungalcell) carrying the two components of the regulatory system of theinvention (e.g., nucleic acid encoding a tTA and a gene of interest,encoding the gene product of interest, linked to a tTA-responsivepromoter) are first grown in a culture medium in the presence oftetracycline or a tetracycline analogue. Under these conditions,expression of the gene of interest is repressed. Next, the concentrationof tetracycline or the tetracycline analogue in the culture medium isreduced to stimulate transcription of the gene of interest. The cellsare then further cultured in the absence of Tc (or analogue thereof)until a desired amount of the gene product encoded by the gene ofinterest is produced by the cells. The gene product can then be isolatedfrom harvested cells or from the culture medium by standard techniques.The invention also provides for large scale production of a protein ofinterest in animals, such as in transgenic farm animals. Advances intransgenic technology have made it possible to produce transgeniclivestock, such as cattle, goats, pigs and sheep (reviewed in Wall, R.J. et al. (1992) J Cell. Biochem. 49:113-120; and Clark, A. J. et al.(1987) Trends in Biotechnology 5:20-24). Accordingly, transgeniclivestock carrying in their genome the components of the regulatorysystem of the invention can be constructed. Thus, by appropriate mating,double transgenic animals carrying a transgene encoding a tTA of theinvention and a transgene comprising a tTA-responsive promoter linked toa gene of interest (the gene of interest may be either an exogenous oran endogenous gene) can be obtained. In the absence of Tc (or analgoue),expression of the gene of interest is stimulated in the transgenicanimals. By administering Tc (or analogue) to the animal, expression ofthe gene of interest can be inhibited. Protein production can betargeted to a particular tissue by linking the nucleic acid encoding thetTA to an appropriate tissue-specific regulatory element(s) which limitsexpression of the transactivator to certain cells. For example, amammary gland-specific regulatory element, such as the milk wheypromoter (U.S. Pat. No. 4,873,316 and European Application PublicationNo. 264,166), can be linked to the tTA-encoding transgene to limitexpression of the transactivator to mammary tissue. Thus, in the absenceof Tc (or analogue), the protein of interest will be produced in themammary tissue of the transgenic animal, whereas protein expression canbe downmodulated by administering Tc or a Tc analogue. The protein canbe designed to be secreted into the milk of the transgenic animal, andif desired, the protein can then be isolated from the milk.

[0172] Having now generally described this invention, the same will beunderstood by reference to the following examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified. The contents of all publications,references, patents and published patent applications cited throughoutthe application are hereby incorporated by reference.

EXAMPLE 1 Regulation of Gene Expression in Cells by tTA

[0173] Materials and Methods

[0174] Construction of the transactivators tTA and tTA_(S). The tetRsequence was originally recovered from pWH510 (Altschmied et al., EMBOJ. 7:4011-4017 (1988), the disclosure of which is fully incorporated byreference herein) by PCR and inserted into pUHD 10-1 (Deustchle et al.,Proc. Natl. Acad. Sci. USA 86:5400-5404 (1989)), resulting in pUHD14-1(see, the Dissertation of Manfred Gossen, “Prokaryotic RepressorOperator Systems in the Control of Eucaryotic Gene Expression,Heidelberg University, 1993, the contents of which are fullyincorporated by reference herein). A unique AflII cleavage site,overlapping the tetR stop codon in this plasmid construct, allows forthe in-frame insertion of coding sequences. To generate tTA, a397-base-pair (bp) MluI/FokI fragment of pMSVP16 (Triezenberg et al.,Genes Dev. 2:718-729 (1988), the disclosure of which is fullyincorporated by reference herein), coding for the C-terminal 130 aminoacids of VP16 of HSV, was blunted by filling in the protruding ends withT4 DNA polymerase. This DNA was inserted in pUHD14-1, previously cleavedwith AflII, and blunted by mung bean nuclease. The resulting plasmid,pUHD15-1, encodes the tTA sequence (FIG. 1, panel a) under the controlof the P_(hCMV) (human cytomegalovirus promoter IE; see below). In ahomologous approach, a DNA fragment coding for the 97-amino acidC-terminal portion of VP 16 was fused to tetR by PCR-mediated cloning.The resulting plasmid, pUHD151-1, encodes the smaller version of thetrans-activator, tTAS (FIG. 1, panel a).

[0175] Construction of P_(hCMV)* and the Luciferase Reporter Plasmid.

[0176] Plasmid pUHC13-1 is a derivative of pUHD10-1 (Deuschle et al.,Proc. Natl. Acad. Sci. USA 86:5400-5404 (1989)). It contains thepromoter-enhancer sequence of P_(hCMV), spanning position +75 toposition −675 (Boshart et al., Cell 41:521-530 (1985)). This promoter isfollowed by a polylinker and the luciferase gene of Photinus pyralisfused to the SV40 small-t intron and poly(A) signal. The latter elementsand the luciferase gene were transferred from pSV2L,AΔ5′ (DeWit et al.,Mol. Cell. Biol. 7:725-737 (1987)). By this transfer, the N-terminus ofluciferase has been modified as described (Deuschle et al., Proc. Natl.Acad. Sci. USA 86:5400-5404 (1989)). The enhancer region of P_(hCMV) wasremoved by PCR-mediated cloning, whereby a Xho I site was introducedadjacent to position −53. The resulting minimal promoter, P_(hCMV)*(FIG. 1, panel b) is part of the reporter plasmid pUHC13-2.

[0177] Construction of P_(hCMV)*-1 and P_(hCMV)*-2.

[0178] To combine P_(hCMV)* with tet operators, the 19-bp invertedrepeat sequence of operator 02 of Tn10 (Triezenberg et al., Genes Dev.2:718-729 (1988)) was synthesized as part of a 42-bp DNA fragment [SEQID NO: 10]:

[0179] (upper strand: 5′ TCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAG 3′).Upon annealing, the two complementary strands exposed the compatibleprotruding ends of a Xho I and a Sal I cleavage site at the 5 and 3′ends, respectively. Ligation of this fragment into the Xho I site of thepolylinker of pT81-luc (Nordeen, S. K., BioTechniques 6:454-457 (1988))created, upon cloning, single as well as multiple inserts of operatorsequences upstream of a thymidine kinase (tk) minimal promoter from HSVcontained in pT81-luc. The tk promoters containing one, two, and sevenoperator sequences were examined for their ability to be activated intransient expression experiments using the HeLa cell line HtTa-l (seebelow). All constructs were active in tTA producing cells in atetracycline-dependent manner. The heptameric version of the tetOsequences caused by far the highest activation of all Ptk-tetOconstructs. It therefore was removed as a Xho1/Sa1 fragment andtransferred into pUHC13-2. Due to the asymmetric location of the tetOwithin the polylinker of pT81-luc, the resulting plasmids pUHC13-3 andpUHC13-4 contain the heptameric tetOs in two orientations differing inthe distance between the operators and position +1 of P_(hCMV) by 19 bp.The two tetO-containing promoters were designated P_(hCMV)*-1 andP_(hCMV)*-2 (FIG. 1, panel b).

[0180] Band-Shift Assay.

[0181] Cytoplasmic and nuclear cell extracts from ˜2×10⁶ cells wereprepared as described by Andrews and Faller, Nucl. Acids Res. 19:2499(1991), except that the cytoplasmic protein fraction was centrifugedonce more (1 hr, 100,000 ×g). Nuclear proteins were extracted by abuffer containing 20 mM Hepes-KOH (pH 7.9), 25% glycerol, 420 mM NaCL,1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mMphenylmethylsulfonyl fluoride. Aliquots (5 μl ) of nuclear extracts weremixed with 15 μl of binding buffer (10 mM Tris HCl, pH 7.5/10 mM MgCl₂)containing 20 μg of calf thymus DNA, 5 μg of bovine serum albumin, and 2fmol of ³²P-labeled tetO DNA. The tetO DNA was isolated from pUHC13-3 asa 42-bp Taq I fragment whose protruding ends were filled in by Klenowenzyme in the presence of [α-³²P)dCTP. After 20 min. at roomtemperature, aliquots of the binding reaction mixture were loaded onto a5% polyacrylamide/0.07% bisacrylamide gel. Electrophoresis was carriedout in 90 mM Tris base/90 mM boric acid/3 mM EDTA at 5 V/cm.

[0182] Luciferase Assays.

[0183] Cell grown to 80% confluency in 35-mm dishes in Eagle's minimumessential medium were washed with 2 ml of phosphate-buffered salinebefore they were lysed in 25 mM Tris phosphate, pH 7.8/2 mMdithiothreitol/2 mM diaminocyclohexanetetraacetic acid/10% glycerol/1%Triton X-100 for 10 min at room temperature. The lysate was scraped offthe culture dishes and centrifuged for 10 sec in an Eppendorfcentrifuge. Next, aliquots (10 pi) of the supernatant were mixed with250 pl of 25 mM glycylglycine/15 mM MgSO₄/5 mM ATP and assayed forluciferase activity in a Lumat LB9501 (Berthold, Wildbad, F. R. G.)using the integral mode (10 sec). D-Luciferin (L6882, Sigma) was used at0.5 mM. The background signal measured in extracts of HeLa cells thatdid not contain a luciferase gene was indistinguishable from theinstrumental background [80-120 relative light units (rlu)/10 sec).Protein content of the lysates was determined according to Bradford(Bradford, M.M., Anal. Biochem.72:248-254 (1976)).

[0184] RESULTS

[0185] Construction and Characterization of the tTA.

[0186] To convert the prokaryotic tet repressor into a eucaryotictransactivator, it was fused to the negatively charged C-terminal domainof HSV-VP16, known to be essential for transactivation. (Triezenberg etal., Genes Dev. 2:718-729 (1988)). Sequences coding for either a 97- ora 127-amino acid C-terminal portion of VP16 were fused to the tetR gene,resulting in the coding sequences of tTAS and tTA, respectively (FIG. 1,panel a). In plasmids coding for tTA (pUHD15-tTAs (pUHD 151-1), thetransactivator sequences are flanked upstream by P_(hCMV) and downstreamby the SV40 poly(A) site. The two fusion proteins did not differ intheir functional in vivo properties. HeLa cells transiently transfectedwith pUHD 15-1 produced a fusion protein of the expected molecular mass(37 kDa), as demonstrated in immunoblots of the electrophoreticallyseparated cytoplasmic and nuclear extracts (FIG. 2, panel a). Whennuclear extracts were mixed with the tetO DNA, the electrophoreticmobility of the DNA was diminished. The specificity of the interactionbetween tTA and operator DNA was confirmed by the finding that nomobility change for tetO DNA was detectable in the presence of thespecific inducer tetracycline (FIG. 2, panel b).

[0187] Construction of a tTA-Dependent Promoter.

[0188] To generate promoters activatable by tTA, tetOs were insertedupstream of minimal promoter sequences. For P_(hCMV), the upstreamenhancer region was removed by PCR and a Xho I cleavage site wasintroduced adjacent to position −53. This minimal promoter, designatedP_(hCMV)*, spans the original P_(hCMV) sequence from +75 to −53 (+1being the first nucleotide transcribed) and, in addition, contains a StuI site around −31 (FIG. 1, panel b). tetO sequences were fused to thiscore promoter by insertions at the Xho I site (FIG. 1). The tetOsequence 02 of Tn10 is a 19-bp inverted repeat to which tetR binds as a46-kDa dimer (Hillen & Wissmann, “Topics in Molecular and StructuralBiology,” in Protein-Nucleic Acid Interaction, Saeger &˜Heinemann, eds.,Macmillan, London, 1989, Vol. 10, pp. 143-162). It was chemicallysynthesized and ligated into the Xho I cleavage site of the polylinkerlocated upstream of the minimal tk promoter in plasmid pT81-luc(Nordeen, S. K., BioTechniques 6:454-457 (1988)). Multiple insertions oftetOs created a set of promoters that contained between 1 and 7 tetOsequences upstream from position −81 of the tk promoter. A Xho 1/Sal Ifragment containing 7 tetOs, fused head to tail, was recovered from oneof the constructs and transferred into the Xho I site upstream ofP_(hCMv)*. Due to the asyrnmetry of the Xho 1/Sal I fragment, twoP_(hCMV)*-tetO constructs were obtained that differ in the distancebetween the operators and position +1 of P_(hCMV), which is 95 bp forP_(hCMV)*-1 and 76 bp for P_(hCMV)*-2. The plasmids containing thesepromoters are designated pUHC13-3 and pUHC13-4, respectively (FIG. 1,panel b). When HeLa cells were transiently transfected with theseplasmids, high levels of luciferase activity were monitored whenever thecells were cotransfected with pUHD15-1, which provided the codingsequence of tTA. Little activity was observed with cultures grown in thepresence of tetracycline (1.0 tg/ml) or with plasmids containingP_(hCMV)* only. Since P_(hCMV)*-1 and P_(hCMV)*-2 were activated by tTAto a significantly higher degree than any of the Ptk constructs, thelatter ones were not investigated further.

[0189] Quantitation of P_(hCMV)*-1 and P_(hCMV)*-2 Activation by tTA.

[0190] To quantify the stimulation of P_(hCMV)*-tetO constructs by tTA,HeLa cell lines were established that contained the P_(hCMV)*-1- or theP_(hCMV)*-2-luciferase, as well as the P_(hCMV)-tTA expression unitsstably integrated. Conditions for culturing and selecting cells havebeen described (Deuschle et al., Proc. Natl. Acad. Sci. USA 86:5400-5405(1989)). In a first step, cells were cotransfected with pUHD15-1 andpSV2neo (Southern &: Berg, J. Mol. Appl. Genet. 1:327-341 (1982)).Clones resistant to G418 were assayed for transactivation of P_(hCMV)*-1by transient transfection with pUHC13-3. In all HeLa cell clones inwhich the tetracycline-responsive promoters were active, tTA was notdetectable by Western blots or by immunofluorescence. Its presence wasjust barely visible in electrophoretic mobility shift experiments ofhighly labeled tetO DNA. This indicates very low intracellularconcentrations of tTA and may reflect a selection against squelchingeffects caused by higher concentrations of VP16-activating domains (Gill& Ptashne, Nature (London) 334:721-724 (1988).

[0191] One of the positive clones, HtTA-1, was then cotransfected with aplasmid carrying the hygromycin-resistance gene (pHMR272; Bernard etal., Exp. Cell Res. 158:237-243 (1985)) and either pUHC13-3 or pUHC13-4,resulting in the X and T series of clones, respectively. Clonesresistant to hygromycin and G418 were assayed for luciferase activity.

[0192] As shown in Table 1 below, in the absence of tetracycline, thisactivity differed in individual clones by almost four orders ofmagnitude. However, in all cases, the luciferase activity was sensitiveto tetracycline in the culture. This demonstrates that the expression ofluciferase is dependent on the function of tTA, which obviously iscapable of activating promoter constructs P_(hCMV)*-1 and P_(hCMV)*-2.TABLE 1 Tetracycline-dependent Luciferase Activity of Different HeLaCell Clones Luciferase activity, rlu/μ of protein Clone With Tc WithoutTc Activation Factor T7 1074 ± 75  79,197 ± 2,119 7.3 × 10¹ T11  2.5 ±0.4 34,695 ± 1,127 1.3 × 10⁴ T12  3.5 ± 0.9 35,298 ± 5,009   1 × 10⁴ T14≦2 33 ± 4 ≧1.5 × 10¹  T15 286 ± 47 49,070 ± 2,784 1.7 × 10² T16 ≦2  541± 133 ≧2.7 × 10²  X1 ≦2 257,081 ± 40,137 ≧2.7 × 10⁵  X2 ≦2 104,840 ±20,833 ≧ 5 × 10⁴ X7 75 ± 7 125,745 ± 18,204 1.6 × 10³ # grown cultures).Luciferase activities of <2 rlu/μg of protein are too close to theinstrumental background to be quantified.

[0193] When the luciferase activity within various clones was monitoredin the presence and absence of tetracycline hydrochloride (Sigma), tworemarkable results emerged. (i) In all clones tested, tTA greatlystimulated promoter activity, even up to five orders of magnitude inclone X1. (ii) In clones T14, T16, X1 and X2 (Table 1), tetracyclinereduced luciferase activity to values that cannot be quantified even athigh protein concentration of extracts due to instrumental limitations(i.e., rlu/μg of protein>2). T his demonstrates that P_(hCMV)*-1 andP_(hCMV)*-2 are virtually silent when integrated in the proper genomicenvironment and that their activity depends exclusively on the action oftTA.

[0194] The tTA inactivation studies were carried out with 1 μg oftetracycline per ml in the culture medium. A partial inactivation of tTAis, however, readily achieved with tetracycline concentrations below 0.1μg/ml, as shown in FIG. 3, panel a. In the two clones analyzed (T12 andX1), a stepwise reduction of the tetracycline concentration in themedium gradually increased the luciferase activity. These results againdemonstrate that, in the case of clone X1, tTA can regulatetranscriptional activity, as monitored by luciferase activity, by overfive orders of magnitude. Moreover, at tetracycline concentrationssufficient for full inactivation of tTA (0.1 μg/ml), no change in growthbehavior or morphology of HeLa cells occurs. Only at tetracyclineconcentrations well above 10 μg/ml were such changes observed uponprolonged incubation.

[0195] Kinetics of Tetracycline Action.

[0196] The time course of tetracycline action was analyzed in culturesgrown in the absence or presence of tetracycline. At time 0, theantibiotic was added to the tetracycline-free cultures (finalconcentration, 1 μg/ml), whereas the tetracycline-containing cultureswere rinsed and incubated in fresh antibiotic-free medium (FIG. 3 panelb). At various times, cells were harvested and analyzed for luciferaseactivity. As shown in FIG. 3 panel b, the depletion of tetracyclineleads to a rapid induction of luciferase activity reaching>20% of thefully induced level within 12 hr. A similarly rapid reduction ofluciferase activity was observed when tetracycline was added to thefully active tetracycline-free system: within 8 hr activity dropped toabout 10% and reached <2% of its original value after 12 hr.

[0197] The fusion of the Tn10-derived E. coli tetR with the activationdomain of VP16 from HSV has generated a transactivator exhibiting all ofthe properties required for the specific and stringent regulation of anindividual gene in a eucaryotic cell. The transactivator tTA produced inHeLa cells binds specifically to tetO sequences in vitro. Thisassociation is prevented by tetracycline. When bound to tetOs placedupstream of minimal promoters, tTA efficiently activates transcriptionfrom such promoters in vivo in a tetracycline-dependent manner. Thetransactivator is produced in HeLa cells in amounts sufficiently highfor strong activation of transcription though low enough to avoid anydetectable squelching effects (Gill & Ptashne Nature (London)334:721-724 (1988)).

[0198] The usefulness of heterologous regulatory systems as the onedescribed here depends decisively on quantitative parameters such as theextent of inactivation and the efficiency of activation of geneexpression as well as the kinetics of transition from one state to theother. For the tet system, these parameters were measured in HeLa celllines that constitutively express tTA and that also contain theluciferase gene stably integrated and under the control of tTA-dependentpromoters. The clones characterized thus far express the luciferase geneto various extents. This is not surprising since differences in theintegration sites and in the number of integrated transcription unitswould be expected. However, in all cases, the expression of luciferaseis sensitive to tetracycline. In some clones, tetracycline has the mostdramatic effect of reducing the luciferase activity from high levelsover several orders of magnitude to background. This demonstrates thatin HeLa cells, the two promoters P_(hCMV)*-1 and P_(hCMV)*-2, have nomeasurable intrinsic activity. Their function strictly depends on tTA.The residual luciferase activity observed in some clones in the presenceof tetracycline must therefore be due to position effects.

[0199] The tTA-dependent promoters can be kept in a partially activatedstate by low concentrations of tetracycline. As shown in FIG. 3 panel a,varying the tetracycline concentration between 0 and 0.1 μg/ml allowsadjustment of promoter activity within a range of several orders ofmagnitude. This may allow assessment also of quantitative parameters ofgene function in vivo.

[0200] The activation and inactivation of tTA by the antibiotic appearsto be not only an efficient but also a rapid process. When cells fromtetracycline containing medium are shifted to tetracycline-free medium,significant luciferase activity is induced within 4 hr and >20% of thesteady-state level is reached within 12 hr after the shift.Interestingly, even the cultures that were only exposed totetracycline-free medium during the washing procedure beforereincubation in tetracycline-containing medium show a small butreproducible increase in luclferase activity that is still detectableafter 4 hr (FIG. 3b).

[0201] When tetracycline is added to a culture of X1 cells, luciferaseactivity is reduced 10-fold within 8 hr and >50 fold within 12 hr. Thisdecrease is remarkably fast if one takes into account the half-life ofluciferase of around 3 hr reported for eucaryotic cells (measured bycycloheximide inhibition: Ilguyen et al., J. Biol. Chem. 264:10487-10492(1989); Thompson et al., Gene 103:171-177 (1991)) and indicates a rapiduptake of tetracycline by HeLa cells followed by a fast and efficientshutdown of transcription. Although the half-life of luciferase and itsmRNA remains to be determined in this system, these conclusions aresupported by observations in plant cells, where tetracycline inactivatestetR within <30 min (Gatz et al., Mol. Gen. Genet 227:229-237 (1991)).

[0202] Taken together, these data show that tetracycline, unlike IPTG ina eucaryotic lacR/O-based system, is able to act fast in cultures ofeucaryotic cells. The possibility of rapidly switching the activity of atTA-dependent promoter not only is of interest in studying gene functionitself but also should allow analysis of mRNA decay rates of individualgenes under physiological conditions.

[0203] In clone X1, tetracycline reduces luciferase activityreproducibly by five orders of magnitude. This suggests that binding oftetracycline to tTA may lower the association constant between thetransactivator and its operator to a much greater extent than thatmeasured for tetR (Takahasi et al., J. Mol. Biol. 187:341-348 (1986))and as described for IPTG in the lacR/O system, where the bindingconstant kRo is reduced only 1000-fold by the inducer (Barkley andBourgeois in The Operon, Miller and Reznikoff (eds.), Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1980; pp. 177-220.)

[0204] On the other hand, the results obtained in transient experimentswith minimal tk promoters fused to single, dimeric, and heptameric tetOsequences strongly suggest a synergistic effect of multiple tTA bindingsites. The efficient inactivation of tTA by tetracycline is thereforemost likely due to a large difference in the binding constants of tTAand tTA/tetracycline for the tetO and the nonlinear effect oftetracycline interfering with a cooperative process.

[0205] In conclusion, the results indicate that promoter-activatingsystems as described here are most promising for regulating individualgenes in higher eucaryotic cells for several reasons. (i) Foractivators, in particular when acting through a cooperative mechanism,intracellular concentrations can be kept low, ensuring an efficientinactivation by the effector—in this case, tetracycline. By contrast,repressors in general complete directly with transcription factorsand/or RNA polymerases for binding within a promoter region. In theabsence of cooperativity, however, the window at which the repressorconcentration is sufficiently high for tight expression but still lowenough for efficient induction may be narrow and not easily adjustablein different systems. (ii) In an activating system, as described here,the synthesis of tTA can be driven by a tissue-specific promoter,whereas the tTAdependent promoters are expected to function tissueindependently, since they may require only general transcription factorsin addition to tTA. By contrast, in a repressor-based system in whichoperators have to be placed within the context of a promoter sequence,an influence on promoter specificity cannot be excluded. (iii) The tetsystem offers specific advantages when compared to the intensely studiedlac system. For example, tetR binds tetracycline much tighter (ka ˜109M⁻¹; Takahashi et al., J. Mol. Biol. 187:341-348 (1986)) than lacRcomplexes IPTG (ka 10⁶ M⁻¹; Barkley & Bourgeois in The Operon, Miller &Rezinkoff, eds., Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.,1980, pp. 177-220).

[0206] Thus, very low, nontoxic concentrations of tetracycline functioneffectively. Moreover, a large number of tetracycline analogues areknown, of which some appear to have far superior properties as effectorsthan tetracycline itself. In this context, it is interesting to notethat detailed information on the pharmacological properties oftetracycline, in particular pharmacokinetic parameters, is available,which will facilitate application of this system in transgenic animals.

EXAMPLE 2

[0207] Regulation of Gene Expression in Transgenic Animals by tTA

[0208] To examine the ability of tTA to regulate gene expression invivo, transgenic strains of mice were constructed which containedheterologous chromosomal insertions of either a tTA expression unit or atTA-responsive promoter operably linked to a reporter gene. Singletransgenic strains containing either the tTA expression unit or thetTA-responsive reporter unit were then cross bred and double transgenicprogeny were identified. The double transgenic animals were thencharacterized as to the ability of tTA, in a tetracycline dependentmanner, to regulate expression of the reporter gene. This exampledemonstrates that tTA effectively stimulates the expression of a geneoperably linked to a tTA responsive promoter in multiple tissues of theanimals in vivo in the absence of tetracycline (or analogue), whereasexpression of the tTA-responsive gene is effectively inhibited inmultiple tissues of the animals when tetracycline or an analogue thereofis administered to the animals. These results demonstrate that thetetracycline-controlled transcriptional regulatory system describedherein functions effectively in animals, in addition to cell lines invitro.

[0209] Generation of mice transgenic for a P_(hCMV)-tTA expression unit

[0210] Mice expressing tTA protein were obtained by pronuclear injectioninto fertilized oocytes of a 2.7kb XhoI-PfmI fragment excised fromplasmid pUHG15-1. This DNA fragment contained the tTA gene (shown in SEQID NO: 1) under the transcriptional control of the human CMV IE promoter(position +75 to −675) together with a rabbit β-globin polyadenylationsite including an intron. The human CMV IE promoter is a constitutivepromoter that allows expression of the tetR-VP16 fusion protein in manycell lines where chromosomal integration of the DNA sequence encodingtTA has occurred and is known to be functional in a variety of tissuesin transgenic mice. DNA was injected into fertilized oocytes at aconcentration of approximately 5 ng per μ1 by standard techniques.Transgenic mice were generated from the injected fertilized oocytesaccording to standard procedures. Transgenic founder mice were analyzedusing polymerase chain reaction (PCR) and Southern hybridization todetect the presence of the tTA transgene in chromosomal DNA of the mice.

[0211] Generation of mice transgenic for the P_(hCMV)*-1 luciferasereporter unit

[0212] Mice carrying a P_(hCMV)*-1 luc reporter gene expression unitwere generated by pronuclear injection into fertilized oocytes of a 3.1kb XhoI-EaeI fragment excised from plasmid pUHC13-3. This DNA-fragmentcontains the luciferase gene under transcriptional control of thetetracycline-responsive P_(hCMV-1) promoter (SEQ ID NO: 5), togetherwith a SV40 t early polyadenylation site including an intron. DNA wasinjected into oocytes at a concentration of approximately 5 ng per μland transgenic mice were generated according to standard procedures.Transgenic founder mice were analyzed using Southern hybridization todetect the presence of the P_(hCMV*-1) luc transgene in chromosomal DNAof the mice.

[0213] Generation of Mice Transgenic for the P_(hCMV*-1) luc andP_(hCMV)tTA

[0214] Having constructed single transgenic mice expressing tTA orcarrying P_(hCMV*-1) luc, double transgenic mice carrying both the tTAexpression vector and the luciferase reporter-units were obtainedthrough cross breeding of heterozygous mice transgenic for one of thetwo transgenes. Double transgenic animals were identified by standardscreenings (e.g., PCR and/or Southern hybridization) to detect thepresence of both the tTA transgene and the P_(hCMV*-)1 luc transgene inchromosomal DNA of the mice.

[0215] Induction and Analysis of Luciferase Activity in Tissue Samplesfrom Mice

[0216] For oral administration, tetracycline or its derivativedoxycycline were given in the drinking water at a concentration of 200μg per ml with 5% sucrose to hide the bitter taste of the antibiotics.For lactating mice, the concentration was 2 mg per ml with 10% sucroseto ensure a sufficient uptake via the milk by the young.

[0217] To analyze luciferase activity, mice were killed by cervicaldislocation and tissue samples were homogenized in 2 ml tubes containing500 μl lysis-buffer (25 mM Tris phosphate, pH 7.8/2 mM DTT/2 mM EDTA/10%glycerol/ 1% Triton X100) using a Ultra-Turrax. The homogenate wasfrozen in liquid nitrogen and centrifuged after thawing for 5 min at15,000g. 2-20 μl of the supernatant were mixed with 250 μl luciferaseassay buffer (25 mM glycylglycine, pH 7.5/ 15 mM MgSO4/ 5 mM ATP) andluciferase activity was measured for 10 sec after the injection of 100μl of a 125 μM luciferin solution using Berthold Lumat LB9501. Theprotein concentration of the homogenate was determined using Bradfordassay and luciferase activity was calculated as relative light units(rlu) per μg of total protein.

[0218] Results

[0219] Mice from 4 lines carrying the P_(hCMV)-tTA transgene (CT1through CT4) were mated with mice from line L7, transgenic forP_(hCMV*-1) luc. This line shows a very low but significant backgroundof luciferase activity in different organs that is probably due toposition effects at the integration side. The luciferase activity indifferent tissues of the double transgenic mice, either in the presenceor absence of the tetracycline analogue doxycycline, is illustratedgraphically in FIG. 14. High luciferase activity was detectable in fivetissues of the double transgenic mice examined: heart, muscle, pancreas,thymus and tongue. The tissue pattern of activated luciferase levels(i.e., in the absence of doxycycline) in the double transgenic mice wassimilar to expression patterns of the hCMV IE promoter reported in theliterature. This is consistent with expression of the luc reporter genebeing regulated by tTA (which is expressed in the mice under the controlof the hCMV IE promoter). After administration of doxycycline to themice for 7 days, luciferase activity was reduced close to backgroundlevels observed in single transgenic mice carrying only the P_(hCMV*-1)luc reporter unit (i.e., the L7 line). Depending on the individualanimals used for comparison of induced and non-induced luciferase level,regulation factors up to 10,000 fold can be estimated e.g. in thepancreas. These results indicate that the tetracycline-controlledtranscripional regulatory system described herein can be used toefficiently regulate expression of genes in transgenic animals.

[0220] EQUIVALENTS

[0221] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

1 10 1008 base pairs nucleic acid double linear DNA (genomic) HerpesSimplex Virus K12, KOS tTA transactivator exon 1..1008 mRNA 1..1008misc. binding 1..207 misc. binding 208..335 CDS 1..1005 1 ATG TCT AGATTA GAT AAA AGT AAA GTG ATT AAC AGC GCA TTA GAG CTG 48 Met Ser Arg LeuAsp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu 1 5 10 15 CTT AAT GAGGTC GGA ATC GAA GGT TTA ACA ACC CGT AAA CTC GCC CAG 96 Leu Asn Glu ValGly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln 20 25 30 AAG CTA GGT GTAGAG CAG CCT ACA TTG TAT TGG CAT GTA AAA AAT AAG 144 Lys Leu Gly Val GluGln Pro Thr Leu Tyr Trp His Val Lys Asn Lys 35 40 45 CGG GCT TTG CTC GACGCC TTA GCC ATT GAG ATG TTA GAT AGG CAC CAT 192 Arg Ala Leu Leu Asp AlaLeu Ala Ile Glu Met Leu Asp Arg His His 50 55 60 ACT CAC TTT TGC CCT TTAGAA GGG GAA AGC TGG CAA GAT TTT TTA CGT 240 Thr His Phe Cys Pro Leu GluGly Glu Ser Trp Gln Asp Phe Leu Arg 65 70 75 80 AAT AAG GCT AAA AGT TTTAGA TGT GCT TTA CTA AGT CAT CGC GAT GGA 288 Asn Lys Ala Lys Ser Phe ArgCys Ala Leu Leu Ser His Arg Asp Gly 85 90 95 GCA AAA GTA CAT TTA GGT ACACGG CCT ACA GAA AAA CAG TAT GAA ACT 336 Ala Lys Val His Leu Gly Thr ArgPro Thr Glu Lys Gln Tyr Glu Thr 100 105 110 CTC GAA AAT CAA TTA GCC TTTTTA TGC CAA CAA GGT TTT TCA CTA GAG 384 Leu Glu Asn Gln Leu Ala Phe LeuCys Gln Gln Gly Phe Ser Leu Glu 115 120 125 AAT GCA TTA TAT GCA CTC AGCGCT GTG GGG CAT TTT ACT TTA GGT TGC 432 Asn Ala Leu Tyr Ala Leu Ser AlaVal Gly His Phe Thr Leu Gly Cys 130 135 140 GTA TTG GAA GAT CAA GAG CATCAA GTC GCT AAA GAA GAA AGG GAA ACA 480 Val Leu Glu Asp Gln Glu His GlnVal Ala Lys Glu Glu Arg Glu Thr 145 150 155 160 CCT ACT ACT GAT AGT ATGCCG CCA TTA TTA CGA CAA GCT ATC GAA TTA 528 Pro Thr Thr Asp Ser Met ProPro Leu Leu Arg Gln Ala Ile Glu Leu 165 170 175 TTT GAT CAC CAA GGT GCAGAG CCA GCC TTC TTA TTC GGC CTT GAA TTG 576 Phe Asp His Gln Gly Ala GluPro Ala Phe Leu Phe Gly Leu Glu Leu 180 185 190 ATC ATA TGC GGA TTA GAAAAA CAA CTT AAA TGT GAA AGT GGG TCC GCG 624 Ile Ile Cys Gly Leu Glu LysGln Leu Lys Cys Glu Ser Gly Ser Ala 195 200 205 TAC AGC CGC GCG CGT ACGAAA AAC AAT TAC GGG TCT ACC ATC GAG GGC 672 Tyr Ser Arg Ala Arg Thr LysAsn Asn Tyr Gly Ser Thr Ile Glu Gly 210 215 220 CTG CTC GAT CTC CCG GACGAC GAC GCC CCC GAA GAG GCG GGG CTG GCG 720 Leu Leu Asp Leu Pro Asp AspAsp Ala Pro Glu Glu Ala Gly Leu Ala 225 230 235 240 GCT CCG CGC CTG TCCTTT CTC CCC GCG GGA CAC ACG CGC AGA CTG TCG 768 Ala Pro Arg Leu Ser PheLeu Pro Ala Gly His Thr Arg Arg Leu Ser 245 250 255 ACG GCC CCC CCG ACCGAT GTC AGC CTG GGG GAC GAG CTC CAC TTA GAC 816 Thr Ala Pro Pro Thr AspVal Ser Leu Gly Asp Glu Leu His Leu Asp 260 265 270 GGC GAG GAC GTG GCGATG GCG CAT GCC GAC GCG CTA GAC GAT TTC GAT 864 Gly Glu Asp Val Ala MetAla His Ala Asp Ala Leu Asp Asp Phe Asp 275 280 285 CTG GAC ATG TTG GGGGAC GGG GAT TCC CCG GGT CCG GGA TTT ACC CCC 912 Leu Asp Met Leu Gly AspGly Asp Ser Pro Gly Pro Gly Phe Thr Pro 290 295 300 CAC GAC TCC GCC CCCTAC GGC GCT CTG GAT ATG GCC GAC TTC GAG TTT 960 His Asp Ser Ala Pro TyrGly Ala Leu Asp Met Ala Asp Phe Glu Phe 305 310 315 320 GAG CAG ATG TTTACC GAT CCC CTT GGA ATT GAC GAG TAC GGT GGG TAG 1008 Glu Gln Met Phe ThrAsp Pro Leu Gly Ile Asp Glu Tyr Gly Gly 325 330 335 335 amino acidsamino acid linear protein 2 Met Ser Arg Leu Asp Lys Ser Lys Val Ile AsnSer Ala Leu Glu Leu 1 5 10 15 Leu Asn Glu Val Gly Ile Glu Gly Leu ThrThr Arg Lys Leu Ala Gln 20 25 30 Lys Leu Gly Val Glu Gln Pro Thr Leu TyrTrp His Val Lys Asn Lys 35 40 45 Arg Ala Leu Leu Asp Ala Leu Ala Ile GluMet Leu Asp Arg His His 50 55 60 Thr His Phe Cys Pro Leu Glu Gly Glu SerTrp Gln Asp Phe Leu Arg 65 70 75 80 Asn Lys Ala Lys Ser Phe Arg Cys AlaLeu Leu Ser His Arg Asp Gly 85 90 95 Ala Lys Val His Leu Gly Thr Arg ProThr Glu Lys Gln Tyr Glu Thr 100 105 110 Leu Glu Asn Gln Leu Ala Phe LeuCys Gln Gln Gly Phe Ser Leu Glu 115 120 125 Asn Ala Leu Tyr Ala Leu SerAla Val Gly His Phe Thr Leu Gly Cys 130 135 140 Val Leu Glu Asp Gln GluHis Gln Val Ala Lys Glu Glu Arg Glu Thr 145 150 155 160 Pro Thr Thr AspSer Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu 165 170 175 Phe Asp HisGln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu 180 185 190 Ile IleCys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Ser Ala 195 200 205 TyrSer Arg Ala Arg Thr Lys Asn Asn Tyr Gly Ser Thr Ile Glu Gly 210 215 220Leu Leu Asp Leu Pro Asp Asp Asp Ala Pro Glu Glu Ala Gly Leu Ala 225 230235 240 Ala Pro Arg Leu Ser Phe Leu Pro Ala Gly His Thr Arg Arg Leu Ser245 250 255 Thr Ala Pro Pro Thr Asp Val Ser Leu Gly Asp Glu Leu His LeuAsp 260 265 270 Gly Glu Asp Val Ala Met Ala His Ala Asp Ala Leu Asp AspPhe Asp 275 280 285 Leu Asp Met Leu Gly Asp Gly Asp Ser Pro Gly Pro GlyPhe Thr Pro 290 295 300 His Asp Ser Ala Pro Tyr Gly Ala Leu Asp Met AlaAsp Phe Glu Phe 305 310 315 320 Glu Gln Met Phe Thr Asp Pro Leu Gly IleAsp Glu Tyr Gly Gly 325 330 335 894 base pairs nucleic acid doublelinear DNA (genomic) Herpes Simplex Virus K12, KOS tTAS transactivatorexon 1..894 mRNA 1..894 misc. binding 1..207 misc. binding 208..297 CDS1..891 3 ATG TCT AGA TTA GAT AAA AGT AAA GTG ATT AAC AGC GCA TTA GAG CTG48 Met Ser Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu 1 510 15 CTT AAT GAG GTC GGA ATC GAA GGT TTA ACA ACC CGT AAA CTC GCC CAG 96Leu Asn Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln 20 25 30AAG CTA GGT GTA GAG CAG CCT ACA TTG TAT TGG CAT GTA AAA AAT AAG 144 LysLeu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys 35 40 45 CGGGCT TTG CTC GAC GCC TTA GCC ATT GAG ATG TTA GAT AGG CAC CAT 192 Arg AlaLeu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His 50 55 60 ACT CACTTT TGC CCT TTA GAA GGG GAA AGC TGG CAA GAT TTT TTA CGT 240 Thr His PheCys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg 65 70 75 80 AAT AACGCT AAA AGT TTT AGA TGT GCT TTA CTA AGT CAT CGC GAT GGA 288 Asn Asn AlaLys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly 85 90 95 GCA AAA GTACAT TTA GGT ACA CGG CCT ACA GAA AAA CAG TAT GAA ACT 336 Ala Lys Val HisLeu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr 100 105 110 CTC GAA AATCAA TTA GCC TTT TTA TGC CAA CAA GGT TTT TCA CTA GAG 384 Leu Glu Asn GlnLeu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu 115 120 125 AAT GCA TTATAT GCA CTC AGC GCT GTG GGG CAT TTT ACT TTA GGT TGC 432 Asn Ala Leu TyrAla Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys 130 135 140 GTA TTG GAAGAT CAA GAG CAT CAA GTC GCT AAA GAA GAA AGG GAA ACA 480 Val Leu Glu AspGln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr 145 150 155 160 CCT ACTACT GAT AGT ATG CCG CCA TTA TTA CGA CAA GCT ATC GAA TTA 528 Pro Thr ThrAsp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu 165 170 175 TTT GATCAC CAA GGT GCA GAG CCA GCC TTC TTA TTC GGC CTT GAA TTG 576 Phe Asp HisGln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu 180 185 190 ATC ATATGC GGA TTA GAA AAA CAA CTT AAA TGT GAA AGT GGG TCT GAT 624 Ile Ile CysGly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Ser Asp 195 200 205 CCA TCGATA CAC ACG CGC AGA CTG TCG ACG GCC CCC CCG ACC GAT GTC 672 Pro Ser IleHis Thr Arg Arg Leu Ser Thr Ala Pro Pro Thr Asp Val 210 215 220 AGC CTGGGG GAC GAG CTC CAC TTA GAC GGC GAG GAC GTG GCG ATG GCG 720 Ser Leu GlyAsp Glu Leu His Leu Asp Gly Glu Asp Val Ala Met Ala 225 230 235 240 CATGCC GAC GCG CTA GAC GAT TTC GAT CTG GAC ATG TTG GGG GAC GGG 768 His AlaAsp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Asp Gly 245 250 255 GATTCC CCG GGT CCG GGA TTT ACC CCC CAC GAC TCC GCC CCC TAC GGC 816 Asp SerPro Gly Pro Gly Phe Thr Pro His Asp Ser Ala Pro Tyr Gly 260 265 270 GCTCTG GAT ATG GCC GAC TTC GAG TTT GAG CAG ATG TTT ACC GAT GCC 864 Ala LeuAsp Met Ala Asp Phe Glu Phe Glu Gln Met Phe Thr Asp Ala 275 280 285 CTTGGA ATT GAC GAG TAC GGT GGG TTC TAG 894 Leu Gly Ile Asp Glu Tyr Gly GlyPhe 290 295 297 amino acids amino acid linear protein 4 Met Ser Arg LeuAsp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu 1 5 10 15 Leu Asn GluVal Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln 20 25 30 Lys Leu GlyVal Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys 35 40 45 Arg Ala LeuLeu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His 50 55 60 Thr His PheCys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg 65 70 75 80 Asn AsnAla Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly 85 90 95 Ala LysVal His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr 100 105 110 LeuGlu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu 115 120 125Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys 130 135140 Val Leu Glu Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr 145150 155 160 Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile GluLeu 165 170 175 Phe Asp His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly LeuGlu Leu 180 185 190 Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu SerGly Ser Asp 195 200 205 Pro Ser Ile His Thr Arg Arg Leu Ser Thr Ala ProPro Thr Asp Val 210 215 220 Ser Leu Gly Asp Glu Leu His Leu Asp Gly GluAsp Val Ala Met Ala 225 230 235 240 His Ala Asp Ala Leu Asp Asp Phe AspLeu Asp Met Leu Gly Asp Gly 245 250 255 Asp Ser Pro Gly Pro Gly Phe ThrPro His Asp Ser Ala Pro Tyr Gly 260 265 270 Ala Leu Asp Met Ala Asp PheGlu Phe Glu Gln Met Phe Thr Asp Ala 275 280 285 Leu Gly Ile Asp Glu TyrGly Gly Phe 290 295 450 base pairs nucleic acid double linear DNA(genomic) Human cytomegalovirus K12, Towne mRNA 382..450 5 GAATTCCTCGAGTTTACCAC TCCCTATCAG TGATAGAGAA AAGTGAAAGT CGAGTTTACC 60 ACTCCCTATCAGTGATAGAG AAAAGTGAAA GTCGAGTTTA CCACTCCCTA TCAGTGATAG 120 AGAAAAGTGAAAGTCGAGTT TACCACTCCC TATCAGTGAT AGAGAAAAGT GAAAGTCGAG 180 TTTACCACTCCCTATCAGTG ATAGAGAAAA GTGAAAGTCG AGTTTACCAC TCCCTATCAG 240 TGATAGAGAAAAGTGAAAGT CGAGTTTACC ACTCCCTATC AGTGATAGAG AAAAGTGAAA 300 GTCGAGCTCGGTACCCGGGT CGAGTAGGCG TGTACGGTGG GAGGCCTATA TAAGCAGAGC 360 TCGTTTAGTGAACCGTCAGA TCGCCTGGAG ACGCCATCCA CGCTGTTTTG ACCTCCATAG 420 AAGACACCGGGACCGATCCA GCCTCCGCGG 450 450 base pairs nucleic acid double linear DNA(genomic) Human cytomegalovirus Towne mRNA 382..450 6 GAATTCCTCGACCCGGGTAC CGAGCTCGAC TTTCACTTTT CTCTATCACT GATAGGGAGT 60 GGTAAACTCGACTTTCACTT TTCTCTATCA CTGATAGGGA GTGGTAAACT CGACTTTCAC 120 TTTTCTCTATCACTGATAGG GAGTGGTAAA CTCGACTTTC ACTTTTCTCT ATCACTGATA 180 GGGAGTGGTAAACTCGACTT TCACTTTTCT CTATCACTGA TAGGGAGTGG TAAACTCGAC 240 TTTCACTTTTCTCTATCACT GATAGGGAGT GGTAAACTCG ACTTTCACTT TTCTCTATCA 300 CTGATAGGGAGTGGTAAACT CGAGTAGGCG TGTACGGTGG GAGGCCTATA TAAGCAGAGC 360 TCGTTTAGTGAACCGTCAGA TCGCCTGGAG ACGCCATCCA CGCTGTTTTG ACCTCCATAG 420 AAGACACCGGGACCGATCCA GCCTCCGCGG 450 398 base pairs nucleic acid double linear DNA(genomic) Herpes Simplex Virus KOS 7 GAGCTCGACT TTCACTTTTC TCTATCACTGATAGGGAGTG GTAAACTCGA CTTTCACTTT 60 TCTCTATCAC TGATAGGGAG TGGTAAACTCGACTTTCACT TTTCTCTATC ACTGATAGGG 120 AGTGGTAAAC TCGACTTTCA CTTTTCTCTATCACTGATAG GGAGTGGTAA ACTCGACTTT 180 CACTTTTCTC TATCACTGAT AGGGAGTGGTAAACTCGACT TTCACTTTTC TCTATCACTG 240 ATAGGGAGTG GTAAACTCGA CTTTCACTTTTCTCTATCAC TGATAGGGAG TGGTAAACTC 300 GAGATCCGGC GAATTCGAAC ACGCAGATGCAGTCGGGGCG GCGCGGTCCG AGGTCCACTT 360 CGCATATTAA GGTGACGCGT GTGGCCTCGAACACCGAG 398 6244 base pairs nucleic acid double circular DNA (genomic)Human cytomegalovirus Towne (hCMV) pUHD BGR3 8 CTCGAGTTTA CCACTCCCTATCAGTGATAG AGAAAAGTGA AAGTCGAGTT TACCACTCCC 60 TATCAGTGAT AGAGAAAAGTGAAAGTCGAG TTTACCACTC CCTATCAGTG ATAGAGAAAA 120 GTGAAAGTCG AGTTTACCACTCCCTATCAG TGATAGAGAA AAGTGAAAGT CGAGTTTACC 180 ACTCCCTATC AGTGATAGAGAAAAGTGAAA GTCGAGTTTA CCACTCCCTA TCAGTGATAG 240 AGAAAAGTGA AAGTCGAGTTTACCACTCCC TATCAGTGAT AGAGAAAAGT GAAAGTCGAG 300 CTCGGTACCC GGGTCGAGTAGGCGTGTACG GTGGGAGGCC TATATAAGCA GAGCTCGTTT 360 AGTGAACCGT CAGATCGCCTGGAGACGCCA TCCACGCTGT TTTGACCTCC ATAGAAGACA 420 CCGGGACCGA TCCAGCCTCCGCGGCCCCGA ATTCGAGCTC GGTACCGGGC CCCCCCTCGA 480 GGTCGACGGT ATCGATAAGCTTGATATCGA ATTCCAGGAG GTGGAGATCC GCGGGTCCAG 540 CCAAACCCCA CACCCATTTTCTCCTCCCTC TGCCCCTATA TCCCGGCACC CCCTCCTCCT 600 AGCCCTTTCC CTCCTCCCGAGAGACGGGGG AGGAGAAAAG GGGAGTTCAG GTCGACATGA 660 CTGAGCTGAA GGCAAAGGAACCTCGGGCTC CCCACGTGGC GGGCGGCGCG CCCTCCCCCA 720 CCGAGGTCGG ATCCCAGCTCCTGGGTCGCC CGGACCCTGG CCCCTTCCAG GGGAGCCAGA 780 CCTCAGAGGC CTCGTCTGTAGTCTCCGCCA TCCCCATCTC CCTGGACGGG TTGCTCTTCC 840 CCCGGCCCTG TCAGGGGCAGAACCCCCCAG ACGGGAAGAC GCAGGACCCA CCGTCGTTGT 900 CAGACGTGGA GGGCGCATTTCCTGGAGTCG AAGCCCCGGA GGGGGCAGGA GACAGCAGCT 960 CGAGACCTCC AGAAAAGGACAGCGGCCTGC TGGACAGTGT CCTCGACACG CTCCTGGCGC 1020 CCTCGGGTCC CGGGCAGAGCCACGCCAGCC CTGCCACCTG CGAGGCCATC AGCCCGTGGT 1080 GCCTGTTTGG CCCCGACCTTCCCGAAGACC CCCGGGCTGC CCCCGCTACC AAAGGGGTGT 1140 TGGCCCCGCT CATGAGCCGACCCGAGGACA AGGCAGGCGA CAGCTCTGGG ACGGCAGCGG 1200 CCCACAAGGT GCTGCCCAGGGGACTGTCAC CATCCAGGCA GCTGCTGCTC CCCTCCTCTG 1260 GGAGCCCTCA CTGGCCGGCAGTGAAGCCAT CCCCGCAGCC CGCTGCGGTG CAGGTAGACG 1320 AGGAGGACAG CTCCGAATCCGAGGGCACCG TGGGCCCGCT CCTGAAGGGC CAACCTCGGG 1380 CACTGGGAGG CACGGCGGCCGGAGGAGGAG CTGCCCCCGT CGCGTCTGGA GCGGCCGCAG 1440 GAGGCGTCGC CCTTGTCCCCAAGGAAGATT CTCGCTTCTC GGCGCCCAGG GTCTCCTTGG 1500 CGGAGCAGGA CGCGCCGGTGGCGCCTGGGC GCTCCCCGCT GGCCACCTCG GTGGTGGATT 1560 TCATCCACGT GCCCATCCTGCCTCTCAACC ACGCTTTCCT GGCCACCCGC ACCAGGCAGC 1620 TGCTGGAGGG GGAGAGCTACGACGGCGGGG CCGCGGCCGC CAGCCCCTTC GTCCCGCAGC 1680 GGGGCTCCCC CTCTGCCTCGTCCACCCCTG TGGCGGGCGG CGACTTCCCC GACTGCACCT 1740 ACCCGCCCGA CGCCGAGCCCAAAGATGACG CGTTCCCCCT CTACGGCGAC TTCCAGCCGC 1800 CCGCCCTCAA GATAAAGGAGGAGGAAGAAG CCGCCGAGGC CGCGGCGCGC TCCCCGCGTA 1860 CGTACCTGGT GGCTGGTGCAAACCCCGCCG CCTTCCCGGA CTTCCAGCTG GCAGCGCCGC 1920 CGCCACCCTC GCTGCCGCCTCGAGTGCCCT CGTCCAGACC CGGGGAAGCG GCGGTGGCGG 1980 CCTCCCCAGG CAGTGCCTCCGTCTCCTCCT CGTCCTCGTC GGGGTCGACC CTGGAGTGCA 2040 TCCTGTACAA GGCAGAAGGCGCGCCGCCCC AGCAGGGCCC CTTCGCGCCG CTGCCCTGCA 2100 AGCCTCCGGG CGCCGGCGCCTGCCTGCTCC CGCGGGACGG CCTGCCCTCC ACCTCCGCCT 2160 CGGGCGCAGC CGCCGGGGCCGCCCCTGCGC TCTACCCGAC GCTCGGCCTC AACGGACTCC 2220 CGCAACTCGG CTACCAGGCCGCCGTGCTCA AGGAGGGCCT GCCGCAGGTC TACACGCCCT 2280 ATCTCAACTA CCTGAGGCCGGATTCAGAAG CCAGTCAGAG CCCACAGTAC AGCTTCGAGT 2340 CACTACCTCA GAAGATTTGTTTGATCTGTG GGGATGAAGC ATCAGGCTGT CATTATGGTG 2400 TCCTCACCTG TGGGAGCTGTAAGGTCTTCT TTAAAAGGGC AATGGAAGGG CAGCATAACT 2460 ATTTATGTGC TGGAAGAAATGACTGCATTG TTGATAAAAT CCGCAGGAAA AACTGCCCGG 2520 CGTGTCGCCT TAGAAAGTGCTGTCAAGCTG GCATGGTCCT TGGAGGGCGA AAGTTTAAAA 2580 AGTTCAATAA AGTCAGAGTCATGAGAGCAC TCGATGCTGT TGCTCTCCCA CAGCCAGTGG 2640 GCATTCCAAA TGAAAGCCAACGAATCACTT TTTCTCCAAG TCAAGAGATA CAGTTAATTC 2700 CCCCTCTAAT CAACCTGTTAATGAGCATTG AACCAGATGT GATCTATGCA GGACATGACA 2760 ACACAAAGCC TGATACCTCCAGTTCTTTGC TGACGAGTCT TAATCAACTA GGCGAGCGGC 2820 AACTTCTTTC AGTGGTAAAATGGTCCAAAT CTCTTCCAGG TTTTCGAAAC TTACATATTG 2880 ATGACCAGAT AACTCTCATCCAGTATTCTT GGATGAGTTT AATGGTATTT GGACTAGGAT 2940 GGAGATCCTA CAAACATGTCAGTGGGCAGA TGCTGTATTT TGCACCTGAT CTAATATTAA 3000 ATGAACAGCG GATGAAAGAATCATCATTCT ATTCACTATG CCTTACCATG TGGCAGATAC 3060 CGCAGGAGTT TGTCAAGCTTCAAGTTAGCC AAGAAGAGTT CCTCTGCATG AAAGTATTAC 3120 TACTTCTTAA TACAATTCCTTTGGAAGGAC TAAGAAGTCA AAGCCAGTTT GAAGAGATGA 3180 GATCAAGCTA CATTAGAGAGCTCATCAAGG CAATTGGTTT GAGGCAAAAA GGAGTTGTTT 3240 CCAGCTCACA GCGTTTCTATCAGCTCACAA AACTTCTTGA TAACTTGCAT GATCTTGTCA 3300 AACAACTTCA CCTGTACTGCCTGAATACAT TTATCCAGTC CCGGGCGCTG AGTGTTGAAT 3360 TTCCAGAAAT GATGTCTGAAGTTATTGCTG CACAGTTACC CAAGATATTG GCAGGGATGG 3420 TGAAACCACT TCTCTTTCATAAAAAGTGAA TGTCAATTAT TTTTCAAAGA ATTAAGTGTT 3480 GTGGTATGTC TTTCGTTTTGGTCAGGATTA TGACGTCTCG AGTTTTTATA ATATTCTGAA 3540 AGGGAATTCC TGCAGCCCGGGGGATCCACT AGTTCTAGAG GATCCAGACA TGATAAGATA 3600 CATTGATGAG TTTGGACAAACCACAACTAG AATGCAGTGA AAAAAATGCT TTATTTGTGA 3660 AATTTGTGAT GCTATTGCTTTATTTGTAAC CATTATAAGC TGCAATAAAC AAGTTAACAA 3720 CAACAATTGC ATTCATTTTATGTTTCAGGT TCAGGGGGAG GTGTGGGAGG TTTTTTAAAG 3780 CAAGTAAAAC CTCTACAAATGTGGTATGGC TGATTATGAT CCTGCAAGCC TCGTCGTCTG 3840 GCCGGACCAC GCTATCTGTGCAAGGTCCCC GGACGCGCGC TCCATGAGCA GAGCGCCCGC 3900 CGCCGAGGCA AGACTCGGGCGGCGCCCTGC CCGTCCCACC AGGTCAACAG GCGGTAACCG 3960 GCCTCTTCAT CGGGAATGCGCGCGACCTTC AGCATCGCCG GCATGTCCCC TGGCGGACGG 4020 GAAGTATCAG CTCGACCAAGCTTGGCGAGA TTTTCAGGAG CTAAGGAAGC TAAAATGGAG 4080 AAAAAAATCA CTGGATATACCACCGTTGAT ATATCCCAAT GGCATCGTAA AGAACATTTT 4140 GAGGCATTTC AGTCAGTTGCTCAATGTACC TATAACCAGA CCGTTCAGCT GCATTAATGA 4200 ATCGGCCAAC GCGCGGGGAGAGGCGGTTTG CGTATTGGGC GCTCTTCCGC TTCCTCGCTC 4260 ACTGACTCGC TGCGCTCGGTCGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG 4320 GTAATACGGT TATCCACAGAATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC 4380 CAGCAAAAGG CCAGGAACCGTAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC 4440 CCCCCTGACG AGCATCACAAAAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA 4500 CTATAAAGAT ACCAGGCGTTTCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC 4560 CTGCCGCTTA CCGGATACCTGTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAA 4620 TGCTCACGCT GTAGGTATCTCAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG 4680 CACGAACCCC CCGTTCAGCCCGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC 4740 AACCCGGTAA GACACGACTTATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA 4800 GCGAGGTATG TAGGCGGTGCTACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT 4860 AGAAGGACAG TATTTGGTATCTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT 4920 GGTAGCTCTT GATCCGGCAAACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG 4980 CAGCAGATTA CGCGCAGAAAAAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG 5040 TCTGACGCTC AGTGGAACGAAAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA 5100 AGGATCTTCA CCTAGATCCTTTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA 5160 TATGAGTAAA CTTGGTCTGACAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG 5220 ATCTGTCTAT TTCGTTCATCCATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA 5280 CGGGAGGGCT TACCATCTGGCCCCAGTGCT GCAATGATAC CGCGAGACCC ACGCTCACCG 5340 GCTCCAGATT TATCAGCAATAAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT 5400 GCAACTTTAT CCGCCTCCATCCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT 5460 TCGCCAGTTA ATAGTTTGCGCAACGTTGTT GCCATTGCTA CAGGCATCGT GGTGTCACGC 5520 TCGTCGTTTG GTATGGCTTCATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA 5580 TCCCCCATGT TGTGCAAAAAAGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT 5640 AAGTTGGCCG CAGTGTTATCACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC 5700 ATGCCATCCG TAAGATGCTTTTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA 5760 TAGTGTATGC GGCGACCGAGTTGCTCTTGC CCGGCGTCAA TACGGGATAA TACCGCGCCA 5820 CATAGCAGAA CTTTAAAAGTGCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA 5880 AGGATCTTAC CGCTGTTGAGATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT 5940 TCAGCATCTT TTACTTTCACCAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC 6000 GCAAAAAAGG GAATAAGGGCGACACGGAAA TGTTGAATAC TCATACTCTT CCTTTTTCAA 6060 TATTATTGAA GCATTTATCAGGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT 6120 TAGAAAAATA AACAAATAGGGGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACGTC 6180 TAAGAAACCA TTATTATCATGACATTAACC TATAAAAATA GGCGTATCAC GAGGCCCTTT 6240 CGTC 6244 4963 basepairs nucleic acid double circular DNA (genomic) Human cytomegaloviruspUHD BGR4 9 CTCGAGTTTA CCACTCCCTA TCAGTGATAG AGAAAAGTGA AAGTCGAGTTTACCACTCCC 60 TATCAGTGAT AGAGAAAAGT GAAAGTCGAG TTTACCACTC CCTATCAGTGATAGAGAAAA 120 GTGAAAGTCG AGTTTACCAC TCCCTATCAG TGATAGAGAA AAGTGAAAGTCGAGTTTACC 180 ACTCCCTATC AGTGATAGAG AAAAGTGAAA GTCGAGTTTA CCACTCCCTATCAGTGATAG 240 AGAAAAGTGA AAGTCGAGTT TACCACTCCC TATCAGTGAT AGAGAAAAGTGAAAGTCGAG 300 CTCGGTACCC GGGTCGAGTA GGCGTGTACG GTGGGAGGCC TATATAAGCAGAGCTCGTTT 360 AGTGAACCGT CAGATCGCCT GGAGACGCCA TCCACGCTGT TTTGACCTCCATAGAAGACA 420 CCGGGACCGA TCCAGCCTCC GCGGCCCCGA ATTCCGGCCA CGACCATGACCATGACCCTC 480 CACACCAAAG CATCTGGGAT GGCCCTACTG CATCAGATCC AAGGGAACGAGCTGGAGCCC 540 CTGAACCGTC CGCAGCTCAA GATCCCCCTG GAGCGGCCCC TGGGCGAGGTGTACCTGGAC 600 AGCAGCAAGC CCGCCGTGTA CAACTACCCC GAGGGCGCCG CCTACGAGTTCAACGCCGCG 660 GCCGCCGCCA ACGCGCAGGT CTACGGTCAG ACCGGCCTCC CCTACGGCCCCGGGTCTGAG 720 GCTGCGGCGT TCGGCTCCAA CGGCCTGGGG GGTTTCCCCC CACTCAACAGCGTGTCTCCG 780 AGCCCGCTGA TGCTACTGCA CCCGCCGCCG CAGCTGTCGC CTTTCCTGCAGCCCCACGGC 840 CAGCAGGTGC CCTACTACCT GGAGAACGAG CCCAGCGGCT ACACGGTGCGCGAGGCCGGC 900 CCGCCGGCAT TCTACAGGCC AAATTCAGAT AATCGACGCC AGGGTGGCAGAGAAAGATTG 960 GCCAGTACCA ATGACAAGGG AAGTATGGCT ATGGAATCTG CCAAGGAGACTCGCTACTGT 1020 GCAGTGTGCA ATGACTATGC TTCAGGCTAC CATTATGGAG TCTGGTCCTGTGAGGGCTGC 1080 AAGGCCTTCT TCAAGAGAAG TATTCAAGGA CATAACGACT ATATGTGTCCAGCCACCAAC 1140 CAGTGCACCA TTGATAAAAA CAGGAGGAAG AGCTGCCAGG CCTGCCGGCTCCGCAAATGC 1200 TACGAAGTGG GAATGATGAA AGGTGGGATA CGAAAAGACC GAAGAGGAGGGAGAATGTTG 1260 AAACACAAGC GCCAGAGAGA TGATGGGGAG GGCAGGGGTG AAGTGGGGTCTGCTGGAGAC 1320 ATGAGAGCTG CCAACCTTTG GCCAAGCCCG CTCATGATCA AACGCTCTAAGAAGAACAGC 1380 CTGGCCTTGT CCCTGACGGC CGACCAGATG GTCATGGCCT TGTTGGATGCTGAGCCCCCC 1440 ATACTCTATT CCGAGTATGA TCCTACCAGA CCCTTCAGTG AAGCTTCGATGATGGGCTTA 1500 CTGACCAACC TGGCAGACAG GGAGCTGGTT CACATGATCA ACTGGGCGAAGAGGGTGCCA 1560 GGCTTTGTGG ATTTGACCCT CCATGATCAG GTCCACCTTC TAGAATGTGCCTGGCTAGAG 1620 ATCCTGATGA TTGGTCTCGT CTGGCGCTCC ATGGAGCACC CAGTGAAGCTACTGTTTGCT 1680 CCTAACTTGC TCTTGGACAG GAACCAGGGA AAATGTGTAG AGGGCATGGTGGAGATCTTC 1740 GACATGCTGC TGGCTACATC ATCTCGGTTC CGCATGATGA ATCTGCAGGGAGAGGAGTTT 1800 GTGTGCCTCA AATCTATTAT TTTGCTTAAT TCTGGAGTGT ACACATTTCTGTCCAGCACC 1860 CTGAAGTCTC TGGAAGAGAA GGACCATATC CACCGAGTCC TGGACAAGATCACAGACACT 1920 TTGATCCACC TGATGGCCAA GGCAGGCCTG ACCCTGCAGC AGCAGCACCAGCGGCTGGCC 1980 CAGCTCCTCC TCATCCTCTC CCACATCAGG CACATGAGTA ACAAAGGCATGGAGCATCTG 2040 TACAGCATGA AGTGCAAGAA CGTGGTGCCC CTCTATGACC TGCTGCTGGAGATGCTGGAC 2100 GCCCACCGCC TACATGCGCC CACTAGCCGT GGAGGGGCAT CCGTGGAGGAGACGGACCAA 2160 AGCCACTTGG CCACTGCGGG CTCTACTTCA TCGCATTCCT TGCAAAAGTATTACATCACG 2220 GGGGAGGCAG AGGGTTTCCC TGCCACAGTC TGAGAGCTCC CTGGCGGAATTCGAGCTCGG 2280 TACCCGGGGA TCCTCTAGAG GATCCAGACA TGATAAGATA CATTGATGAGTTTGGACAAA 2340 CCACAACTAG AATGCAGTGA AAAAAATGCT TTATTTGTGA AATTTGTGATGCTATTGCTT 2400 TATTTGTAAC CATTATAAGC TGCAATAAAC AAGTTAACAA CAACAATTGCATTCATTTTA 2460 TGTTTCAGGT TCAGGGGGAG GTGTGGGAGG TTTTTTAAAG CAAGTAAAACCTCTACAAAT 2520 GTGGTATGGC TGATTATGAT CCTGCAAGCC TCGTCGTCTG GCCGGACCACGCTATCTGTG 2580 CAAGGTCCCC GGACGCGCGC TCCATGAGCA GAGCGCCCGC CGCCGAGGCAAGACTCGGGC 2640 GGCGCCCTGC CCGTCCCACC AGGTCAACAG GCGGTAACCG GCCTCTTCATCGGGAATGCG 2700 CGCGACCTTC AGCATCGCCG GCATGTCCCC TGGCGGACGG GAAGTATCAGCTCGACCAAG 2760 CTTGGCGAGA TTTTCAGGAG CTAAGGAAGC TAAAATGGAG AAAAAAATCACTGGATATAC 2820 CACCGTTGAT ATATCCCAAT GGCATCGTAA AGAACATTTT GAGGCATTTCAGTCAGTTGC 2880 TCAATGTACC TATAACCAGA CCGTTCAGCT GCATTAATGA ATCGGCCAACGCGCGGGGAG 2940 AGGCGGTTTG CGTATTGGGC GCTCTTCCGC TTCCTCGCTC ACTGACTCGCTGCGCTCGGT 3000 CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG GTAATACGGTTATCCACAGA 3060 ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC CAGCAAAAGGCCAGGAACCG 3120 TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC CCCCCTGACGAGCATCACAA 3180 AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGATACCAGGCGTT 3240 TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTACCGGATACCT 3300 GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAA TGCTCACGCTGTAGGTATCT 3360 CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG CACGAACCCCCCGTTCAGCC 3420 CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAAGACACGACTT 3480 ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATGTAGGCGGTGC 3540 TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGGACAGTATTTGGTAT 3600 CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTTGATCCGGCAA 3660 ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTACGCGCAGAAA 3720 AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTCAGTGGAACGA 3780 AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCACCTAGATCCT 3840 TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAACTTGGTCTGA 3900 CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTATTTCGTTCATC 3960 CATAGTTGCC TGATCCCCGT CGTGTAGATA ACTACGATAC GGGAGGGCTTACCATCTGGC 4020 CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG CTCCAGATTTATCAGCAATA 4080 AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG CAACTTTATCCGCCTCCATC 4140 CAGTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT CGCCAGTTAATAGTTTGCGC 4200 AACGTTGTTG CCATTGCTAC AGGCATCGTG GTGTCACGCT CGTCGTTTGGTATGGCTTCA 4260 TTCAGCTCCG GTTCCCAACG ATCAAGGCGA GTTACATGAT CCCCCATGTTGTGCAAAAAA 4320 GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA AGTTGGCCGCAGTGTTATCA 4380 CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA TGCCATCCGTAAGATGCTTT 4440 TCTGTGACTG GTGAGTACTC AACCAAGTCA TTCTGAGAAT AGTGTATGCGGCGACCGAGT 4500 TGCTCTTGCC CGGCGTCAAT ACGGGATAAT ACCGCGCCAC ATAGCAGAACTTTAAAAGTG 4560 CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAAGGATCTTACCGCTGTTGAGGA 4620 TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTTCAGCATCTTT TACTTTCACC 4680 AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCGCAAAAAAGGG AATAAGGGCG 4740 ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAATATTATTGAAG CATTTATCAG 4800 GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTTAGAAAAATAA ACAAATAGGG 4860 GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGACGTCTAAGAAACCAT TATTATCATG 4920 ACATTAACCT ATAAAAATAG GCGTATCACG AGGCCCTTTCGTC 4963 42 base pairs nucleic acid double linear 10 TCGAGTTTACCACTCCCTAT CAGTGATAGA GAAAAGTGAA AG 42

1. A method for regulating expression of a tet operator-linked gene in acell of a subject, comprising: introducing into the cell a nucleic acidmolecule encoding a tetracycline-controllable transactivator (tTA), thetTA comprising a Tet repressor operably linked to a polypeptide whichdirectly or indirectly activates transcription in eucaryotic cells; andmodulating the concentration of a tetracycline, or analogue thereof, inthe subject.
 2. The method of claim 1, wherein the Tet repressor of thetTA is a Tn10-derived Tet repressor.
 3. The method of claim 1, whereinthe polypeptide of the tTA which directly or indirectly activatestranscription in eucaryotic cells is from herpes simplex virus virionprotein
 16. 4. The method of claim 1, wherein the nucleic acid moleculeencoding the tTA is integrated randomly in a chromosome of the cell. 5.The method of claim 1, wherein the nucleic acid molecule encoding thetTA is integrated at a predetermined location within a chromosome of thecell.
 6. The method of claim 1, wherein the nucleic acid moleculeencoding the tTA is introduced into the cell ex vivo, the method furthercomprising administering the cell to the subject.
 7. The method of claim1, wherein the tet operator-linked gene is an endogenous gene of thecell which has been operatively linked to the at least one tet operatorsequence.
 8. The method of claim 1, wherein the tet operator-linked geneis an exogenous gene which has been introduced into the cells.
 9. Themethod of claim 1, wherein the tetracycline analogue isanhydrotetracycline, doxycycline or cyanotetracycline.
 10. A method forregulating expression of a gene in a cell of a subject, comprising:obtaining the cell from the subject; introducing into the cell a firstnucleic acid molecule which operatively links a gene to at least one tetoperator sequence; introducing into the cell a second nucleic acidmolecule encoding a tetracycline-controllable transactivator (tTA), thetTA comprising a Tet repressor operably linked to a polypeptide whichdirectly or indirectly activates transcription in eucaryotic cells, toform a modified cell; administering the modified cell to the subject;and modulating the concentration of a tetracycline, or analogue thereof,in the subject.
 11. The method of claim 10, wherein the Tet repressor ofthe tTA is a Tn10-derived Tet repressor.
 12. The method of claim 10,wherein the polypeptide of the tTA which directly or indirectlyactivates transcription in eucaryotic cells is from herpes simplex virusvirion protein
 16. 13. The method of claim 10, wherein the nucleic acidmolecule encoding the tTA is integrated randomly in a chromosome of thecell.
 14. The method of claim 10, wherein the nucleic acid moleculeencoding the tTA is integrated by homologous recombination at apredetermined location within a chromosome of the cell.
 15. The methodof claim 10, wherein the first nucleic acid molecule operatively linksan endogenous gene of the cell to at least one tet operator sequence.16. The method of claim 10, wherein the first nucleic acid moleculecomprises a gene operatively linked to at least one tet operatorsequence.
 17. The method of claim 10, wherein the tetracycline analogueis anhydrotetracycline, doxycycline or cyanotetracycline.